Immunotherapy with a*01 restricted peptides and combination of peptides against cancers and related methods

ABSTRACT

The present invention relates to peptides, proteins, nucleic acids and cells for use in immunotherapeutic methods. In particular, the present invention relates to the immunotherapy of cancer. The present invention furthermore relates to tumor-associated T-cell peptide epitopes, alone or in combination with other tumor-associated peptides that can for example serve as active pharmaceutical ingredients of vaccine compositions that stimulate anti-tumor immune responses, or to stimulate T cells ex vivo and transfer into patients. Peptides bound to molecules of the major histocompatibility complex (MHC), or peptides as such, can also be targets of antibodies, soluble T-cell receptors, and other binding molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a Continuation of U.S. patent applicationSer. No. 16/575,250, filed on 18 Sep. 2019, which claims the benefit ofU.S. Provisional Patent Application No. 62/732,863, filed on 18 Sep.2018 and German Application No. 10 2018 122 900.3, filed on 18 Sep.2018, the contents of which are hereby incorporated by reference intheir entireties.

This application is related to International Application No.PCT/EP2019/075065, filed 18 Sep. 2019.

REFERENCE TO SEQUENCE LISTING SUBMITTED AS A COMPLIANT ASCII TEXT FILE(.txt)

Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (seeMPEP § 2442.03(a)), a Sequence Listing in the form of an ASCII-complianttext file (entitled “Sequence_Listing_2912919-099006_ST25.txt” createdon 30 Jun. 2020, and 68,247 bytes in size) is submitted concurrentlywith the instant application, and the entire contents of the SequenceListing are incorporated herein by reference.

BACKGROUND Field

The present invention relates to peptides, proteins, nucleic acids andcells for use in immunotherapeutic methods. In particular, the presentinvention relates to the immunotherapy of cancer. The present inventionfurthermore relates to tumor-associated T-cell peptide epitopes, aloneor in combination with other tumor-associated peptides that can forexample serve as active pharmaceutical ingredients of vaccinecompositions that stimulate anti-tumor immune responses, or to stimulateT cells ex vivo and transfer into patients. Peptides bound to moleculesof the major histocompatibility complex (MHC), or peptides as such, canalso be targets of antibodies, soluble T-cell receptors, and otherbinding molecules.

The present invention relates to several novel peptide sequences andtheir variants derived from HLA class I molecules of human tumor cellsthat can be used in vaccine compositions for eliciting anti-tumor immuneresponses, or as targets for the development ofpharmaceutically/immunologically active compounds and cells.

DESCRIPTION OF RELATED ART

According to the World Health Organization (WHO), cancer ranged amongthe four major non-communicable deadly diseases worldwide in 2012. Forthe same year, colorectal cancer, breast cancer and respiratory tractcancers were listed within the top 10 causes of death in high incomecountries.

Epidemiology

In 2012, 14.1 million new cancer cases, 32.6 million patients sufferingfrom cancer (within 5 years of diagnosis) and 8.2 million cancer deathswere estimated worldwide (Bray et al., 2013; Ferlay et al., 2013).

Within the groups of brain cancer, leukemia and lung cancer the currentinvention specifically focuses on glioblastoma (GBM), chroniclymphocytic leukemia (CLL) and acute myeloid leukemia (AML), non-smallcell and small cell lung cancer (NSCLC and SCLC), respectively.

GBM is the most common central nervous system malignancy with anage-adjusted incidence rate of 3.19 per 100,000 inhabitants within theUnited States. GBM has a very poor prognosis with a 1-year survival rateof 35% and a 5-year survival rate lower than 5%. Male gender, older ageand ethnicity appear to be risk factors for GBM (Thakkar et al., 2014).

CLL is the most common leukemia in the Western world where it comprisesabout one third of all leukemia. Incidence rates are similar in the USand Europe, and estimated new cases are about 16,000 per year. CLL ismore common in Caucasians than in Africans, rarer in Hispanics andNative Americans and seldom in Asians. In people of Asian origin, CLLincidence rates are 3-fold lower than in Caucasians (Gunawardana et al.,2008). The five-year overall survival for patients with CLL is about79%.

AML is the second most common type of leukemia diagnosed in both adultsand children. Estimated new cases in the United States are about 21,000per year. The five-year survival rate of people with AML isapproximately 25%.

Lung cancer is the most common type of cancer worldwide and the leadingcause of death from cancer in many countries. Lung cancer is subdividedinto small cell lung cancer and non-small cell lung cancer. NSCLCincludes the histological types adenocarcinoma, squamous cell carcinomaand large cell carcinoma and accounts for 85% of all lung cancers in theUnited States. The incidence of NSCLC is closely correlated with smokingprevalence, including current and former smokers and the five-yearsurvival rate was reported to be 15% (Molina et al., 2008; World CancerReport, 2014).

Considering the severe side-effects and expense associated with treatingcancer, there is a need to identify factors that can be used in thetreatment of cancer in general and acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer in particular. Thereis also a need to identify factors representing biomarkers for cancer ingeneral and acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lungcancer (including non-small cell lung cancer adenocarcinoma, squamouscell non-small cell lung cancer, and small cell lung cancer), ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, urinary bladder carcinoma, uterine and endometrialcancer in particular, leading to better diagnosis of cancer, assessmentof prognosis, and prediction of treatment success for people with theA*01 allele.

Immunotherapy of cancer represents an option of specific targeting ofcancer cells while minimizing side effects. Cancer immunotherapy makesuse of the existence of tumor associated antigens.

The current classification of tumor associated antigens (TAAs) comprisesthe following major groups:

a) Cancer-testis antigens: The first TAAs ever identified that can berecognized by T cells belong to this class, which was originally calledcancer-testis (CT) antigens because of the expression of its members inhistologically different human tumors and, among normal tissues, only inspermatocytes/spermatogonia of testis and, occasionally, in placenta.Since the cells of testis do not express class I and II HLA molecules,these antigens cannot be recognized by T cells in normal tissues and cantherefore be considered as immunologically tumor-specific. Well-knownexamples for CT antigens are the MAGE family members and NY-ESO-1.b) Differentiation antigens: These TAAs are shared between tumors andthe normal tissue from which the tumor arose. Most of the knowndifferentiation antigens are found in melanomas and normal melanocytes.Many of these melanocyte lineage-related proteins are involved inbiosynthesis of melanin and are therefore not tumor specific butnevertheless are widely used for cancer immunotherapy. Examples include,but are not limited to, tyrosinase and Melan-A/MART-1 for melanoma orPSA for prostate cancer.c) Over-expressed TAAs: Genes encoding widely expressed TAAs have beendetected in histologically different types of tumors as well as in manynormal tissues, generally with lower expression levels. It is possiblethat many of the epitopes processed and potentially presented by normaltissues are below the threshold level for T-cell recognition, whiletheir over-expression in tumor cells can trigger an anticancer responseby breaking previously established tolerance. Prominent examples forthis class of TAAs are Her-2/neu, survivin, telomerase, or WT1.d) Tumor-specific antigens: These unique TAAs arise from mutations ofnormal genes (such as β-catenin, CDK4, etc.). Some of these molecularchanges are associated with neoplastic transformation and/orprogression. Tumor-specific antigens are generally able to induce strongimmune responses without bearing the risk for autoimmune reactionsagainst normal tissues. On the other hand, these TAAs are in most casesonly relevant to the exact tumor on which they were identified and areusually not shared between many individual tumors. Tumor-specificity (or-association) of a peptide may also arise if the peptide originates froma tumor- (-associated) exon in case of proteins with tumor-specific(-associated) isoforms.e) TAAs arising from abnormal post-translational modifications: SuchTAAs may arise from proteins which are neither specific noroverexpressed in tumors but nevertheless become tumor associated byposttranslational processes primarily active in tumors. Examples forthis class arise from altered glycosylation patterns leading to novelepitopes in tumors as for MUC1 or events like protein splicing duringdegradation which may or may not be tumor specific.f) Oncoviral proteins: These TAAs are viral proteins that may play acritical role in the oncogenic process and, because they are foreign(not of human origin), they can evoke a T-cell response. Examples ofsuch proteins are the human papilloma type 16 virus proteins, E6 and E7,which are expressed in cervical carcinoma.

T-cell based immunotherapy targets peptide epitopes derived fromtumor-associated or tumor-specific proteins, which are presented bymolecules of the major histocompatibility complex (MHC). The antigensthat are recognized by the tumor specific T lymphocytes, that is, theepitopes thereof, can be molecules derived from all protein classes,such as enzymes, receptors, transcription factors, etc. which areexpressed and, as compared to unaltered cells of the same origin,usually up-regulated in cells of the respective tumor.

There are two classes of MHC-molecules, MHC class I and MHC class II.MHC class I molecules are composed of an alpha heavy chain andbeta-2-microglobulin, MHC class II molecules of an alpha and a betachain. Their three-dimensional conformation results in a binding groove,which is used for non-covalent interaction with peptides.

MHC class I molecules can be found on most nucleated cells. They presentpeptides that result from proteolytic cleavage of predominantlyendogenous proteins, defective ribosomal products (DRIPs) and largerpeptides. However, peptides derived from endosomal compartments orexogenous sources are also frequently found on MHC class I molecules.This non-classical way of class I presentation is referred to ascross-presentation in the literature (Brossart and Bevan, 1997; Rock etal., 1990). MHC class II molecules can be found predominantly onprofessional antigen presenting cells (APCs), and primarily presentpeptides of exogenous or transmembrane proteins that are taken up byAPCs e.g. during endocytosis and are subsequently processed.

Complexes of peptide and MHC class I are recognized by CD8-positive Tcells bearing the appropriate T-cell receptor (TCR), whereas complexesof peptide and MHC class II molecules are recognized byCD4-positive-helper-T cells bearing the appropriate TCR. It is wellknown that the TCR, the peptide and the MHC are thereby present in astoichiometric amount of 1:1:1.

CD4-positive helper T cells play an important role in inducing andsustaining effective responses by CD8-positive cytotoxic T cells. Theidentification of CD4-positive T-cell epitopes derived from tumorassociated antigens (TAA) is of great importance for the development ofpharmaceutical products for triggering anti-tumor immune responses(Gnjatic et al., 2003). At the tumor site, T helper cells, support acytotoxic T cell- (CTL-) friendly cytokine milieu (Mortara et al., 2006)and attract effector cells, e.g. CTLs, natural killer (NK) cells,macrophages, and granulocytes (Hwang et al., 2007).

In the absence of inflammation, expression of MHC class II molecules ismainly restricted to cells of the immune system, especially professionalantigen-presenting cells (APC), e.g., monocytes, monocyte-derived cells,macrophages, dendritic cells. In cancer patients, cells of the tumorhave been found to express MHC class II molecules (Dengjel et al.,2006).

Longer (elongated) peptides of the invention can act as MHC class IIactive epitopes.

T-helper cells, activated by MHC class II epitopes, play an importantrole in orchestrating the effector function of CTLs in anti-tumorimmunity. T-helper cell epitopes that trigger a T-helper cell responseof the TH1 type support effector functions of CD8-positive killer Tcells, which include cytotoxic functions directed against tumor cellsdisplaying tumor-associated peptide/MHC complexes on their cellsurfaces. In this way tumor-associated T-helper cell peptide epitopes,alone or in combination with other tumor-associated peptides, can serveas active pharmaceutical ingredients of vaccine compositions thatstimulate anti-tumor immune responses.

It was shown in mammalian animal models, e.g., mice, that even in theabsence of CD8-positive T lymphocytes, CD4-positive T cells aresufficient for inhibiting manifestation of tumors via inhibition ofangiogenesis by secretion of interferon-gamma (IFNγ) (Beatty andPaterson, 2001; Mumberg et al., 1999). There is evidence for CD4 T cellsas direct anti-tumor effectors (Braumuller et al., 2013; Tran et al.,2014).

Since the constitutive expression of HLA class II molecules is usuallylimited to immune cells, the possibility of isolating class II peptidesdirectly from primary tumors was previously not considered possible.However, Dengjel et al. were successful in identifying a number of MHCClass II epitopes directly from tumors (WO 2007/028574, EP 1 760 088B1).

Since both types of response, CD8 and CD4 dependent, contribute jointlyand synergistically to the anti-tumor effect, the identification andcharacterization of tumor-associated antigens recognized by either CD8+T cells (ligand: MHC class I molecule+peptide epitope) or byCD4-positive T-helper cells (ligand: MHC class II molecule+peptideepitope) is important in the development of tumor vaccines.

For an MHC class I peptide to trigger (elicit) a cellular immuneresponse, it also must bind to an MHC-molecule. This process isdependent on the allele of the MHC-molecule and specific polymorphismsof the amino acid sequence of the peptide. MHC-class-1-binding peptidesare usually 8-12 amino acid residues in length and usually contain twoconserved residues (“anchors”) in their sequence that interact with thecorresponding binding groove of the MHC-molecule. In this way each MHCallele has a “binding motif” determining which peptides can bindspecifically to the binding groove.

In the MHC class I dependent immune reaction, peptides not only have tobe able to bind to certain MHC class I molecules expressed by tumorcells, they subsequently also have to be recognized by T cells bearingspecific T cell receptors (TCR).

For proteins to be recognized by T-lymphocytes as tumor-specific or-associated antigens, and to be used in a therapy, particularprerequisites must be fulfilled. The antigen should be expressed mainlyby tumor cells and not, or in comparably small amounts, by normalhealthy tissues. In a preferred embodiment, the peptide should beover-presented by tumor cells as compared to normal healthy tissues. Itis furthermore desirable that the respective antigen is not only presentin a type of tumor, but also in high concentrations (i.e. copy numbersof the respective peptide per cell). Tumor-specific and tumor-associatedantigens are often derived from proteins directly involved intransformation of a normal cell to a tumor cell due to their function,e.g. in cell cycle control or suppression of apoptosis. Additionally,downstream targets of the proteins directly causative for atransformation may be up-regulated and thus may be indirectlytumor-associated.

Such indirect tumor-associated antigens may also be targets of avaccination approach (Singh-Jasuja et al., 2004). It is essential thatepitopes are present in the amino acid sequence of the antigen, in orderto ensure that such a peptide (“immunogenic peptide”), being derivedfrom a tumor associated antigen, leads to an in vitro or in vivoT-cell-response.

Basically, any peptide able to bind an MHC molecule may function as aT-cell epitope. A prerequisite for the induction of an in vitro or invivo T-cell-response is the presence of a T cell having a correspondingTCR and the absence of immunological tolerance for this particularepitope.

Therefore, TAAs are a starting point for the development of a Tcell-based therapy including but not limited to tumor vaccines. Themethods for identifying and characterizing the TAAs are usually based onthe use of T-cells that can be isolated from patients or healthysubjects, or they are based on the generation of differentialtranscription profiles or differential peptide expression patternsbetween tumors and normal tissues. However, the identification of genesover-expressed in tumor tissues or human tumor cell lines, orselectively expressed in such tissues or cell lines, does not provideprecise information as to the use of the antigens being transcribed fromthese genes in an immune therapy. This is because only an individualsubpopulation of epitopes of these antigens are suitable for such anapplication since a T cell with a corresponding TCR has to be presentand the immunological tolerance for this particular epitope needs to beabsent or minimal. In a very preferred embodiment of the invention it istherefore important to select only those over- or selectively presentedpeptides against which a functional and/or a proliferating T cell can befound. Such a functional T cell is defined as a T cell, which uponstimulation with a specific antigen can be clonally expanded and is ableto execute effector functions (“effector T cell”).

In case of targeting peptide-MHC by specific TCRs (e.g. soluble TCRs)and antibodies or other binding molecules (scaffolds) according to theinvention, the immunogenicity of the underlying peptides is secondary.In these cases, the presentation is the determining factor.

BRIEF SUMMARY OF THE INVENTION

In a first aspect of the present invention, the present inventionrelates to a peptide comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 1 to SEQ ID NO: 398 or a variant sequencethereof which is at least 77%, preferably at least 88%, homologous(preferably at least 77% or at least 88% identical) to SEQ ID NO: 1 toSEQ ID NO: 398, wherein said variant binds to MHC and/or induces T cellscross-reacting with said peptide, or a pharmaceutical acceptable saltthereof, wherein said peptide is not the underlying full-lengthpolypeptide.

The present invention further relates to a peptide of the presentinvention comprising a sequence that is selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 398 or a variant thereof, whichis at least 77%, preferably at least 88%, homologous (preferably atleast 77% or at least 88% identical) to SEQ ID NO: 1 to SEQ ID NO: 398,wherein said peptide or variant thereof has an overall length of between8 and 100, preferably between 8 and 30, and most preferred of between 8and 14 amino acids.

The following tables show the peptides according to the presentinvention, their respective SEQ ID NOs, and the prospective source(underlying) genes for these peptides. In Table 1, peptides with SEQ IDNO: 1 to SEQ ID NO: 385 bind to HLA-A*01. The peptides in Table havebeen disclosed before in large listings as results of high-throughputscreenings with high error rates or calculated using algorithms but havenot been associated with cancer at all before. In Table 2, peptides withSEQ ID NO: 386 to SEQ ID NO: 398 bind to HLA-A*01. The peptides in Tableare additional peptides that may be useful in combination with the otherpeptides of the invention. In Table 3, peptides with SEQ ID NO: 399 toSEQ ID NO: 431 bind to HLA-A*01.

TABLE 1 Peptides according to the present invention. SEQ HLA ID allo-No. Sequence Gene(s) type 1 TLDSTRTLY TRPM8 A*01 2 VDPIGHLY MAGEA3 A*013 FGTTPAAEYF SLC6A3 A*01 4 RIEAIRAEY TRIM51, TRIM51EP, TRIM51GP, A*01TRIM51HP 5 FMVIAGMPLFY SLC6A3 A*01 6 ARDPITFSF UMODL1 A*01 7 ASDDVRIEVGLUMODL1 A*01 Y 8 TSRAANIPGY C7orf72 A*01 9 QLDSTLDSY CYP4Z1, CYP4Z2P A*0110 VSERTGISY ITIH6 A*01 11 ASDHWRGRY SLC45A3 A*01 12 YTDFVGEGLY SLC45A3A*01 13 NTHTGTRPY CTCFL A*01 14 QSEKEPGQQY RHOXF2, RHOXF2B A*01 15YLDSSKPAVY ESR1 A*01 16 NSDISIPEY SCGB2A1 A*01 17 ASWAVLCYY ANO7 A*01 18RSDPVSLRY COL20A1 A*01 19 LTEGHSGNY FCRL5 A*01 20 LSAQHRMLA EML6 A*01 21LSSAVNPIIY NMUR2 A*01 22 VMDTLGLFY TRPM8 A*01 23 DTDPLKAAGL SOX14 A*0124 NLDHYTNAY GLYATL3 A*01 25 AMMQEAQLAY SOX1 A*01 26 ASDDFRSKYKRT34, LOC100653049 A*01 27 PSEVPVDSHY LOXL4 A*01 28 PSEVPVDSHYY LOXL4A*01 29 TLEDLDNLYNY EPYC A*01 30 VTTDKPRAY ITIH6 A*01 31 VSDHLQAGMLHEPHL1 A*01 GQY 32 GTDKQNSTLRY HMCN1 A*01 33 SMDPVTGYQY PAX3 A*01 34SSWSAGENDS HRNR A*01 Y 35 SWSAGENDSY HRNR A*01 S 36 MTSTEQSLY GREB1 A*0137 MTSTEQSLYY GREB1 A*01 38 KSWSQSSSLM F5 A*01 Y 39 WSQSSSLMY F5 A*01 40TSDQLGYSY DCT A*01 41 HSDLLEDSKY NAT1 A*01 42 ASDVDTLLK PTPRZ1 A*01 43ETEPERHLGSY NKX3-1 A*01 44 IPSFNEMVY PTPRZ1 A*01 45 NLDPNKIY STK31 A*0146 RSDPGGGGLA MEX3B A*01 YAAY 47 WSDGVPLLY BCAN A*01 48 FTTQDELLVY SEMG2A*01 49 GSFSIQHTY SEMG2 A*01 50 ITDEDEDMLSY TSPY1, TSPY10, TSPY2, TSPY3,A*01 TSPY4, TSPY8, TSPY9P 51 STEERRLNY SEMG2 A*01 52 TTQDELLVY SEMG2A*01 53 YLEDRPLSQLY LOXL4 A*01 54 EVDIHTIHY HEPHL1 A*01 55 ATEGDVLNYCOL19A1 A*01 56 VTEYAEEIYQY CCNA1 A*01 57 ASDPASSTSCY CYP1A1 A*01 58YLENSASWY DLX5 A*01 59 FTDSQGNDIK SLC45A2 A*01 60 MTEKFLFLYERVV-1, ERVV-2 A*01 61 SSDIVALGGFL KLHDC7B A*01 Y 62 VSELVTTGHY VCANA*01 63 TSEISQNALMY ROS1 A*01 64 TSEISQNALMY ROS1 A*01 Y 65 SSDFDPLVYRNF43, SUPT4H1 A*01 66 IATVIQLFY HAS2, HAS3 A*01 67 NVDQNQNSY HMCN1 A*0168 QSLPEFGLTY FERMT1 A*01 69 QSLPEFGLTYY FERMT1 A*01 70 YTELVEEKY CAPN6A*01 71 LTDSTTRTTY MUC3A A*01 72 VTDSTTKIAY LOC101060740 A*01 73STDSASYY APOB A*01 74 EMEQQSQEY KRT13, KRT16 A*01 75 FTDYELKAY ABCB11A*01 76 QTDVERIKDTY LAMA3 A*01 77 FTSDTGLEY DNMT3B A*01 78 QLDSAVKNLYZNF215 A*01 79 ASDLEPRELLS PCDHGB1 A*01 Y 80 ELCPLPGTSAY FBN3 A*01 81YSDLHTPGRY PTCHD4 A*01 82 LTEKSHIRY C8orf48 A*01 83 DTEFHGGLHYLOC100124692 A*01 84 ESEMIKFASYY ABCB11 A*01 85 SSDNYEHWLY SFMBT1 A*0186 VDPASNTY MAGEA4 A*01 87 AFDDIATYF SSX1 A*01/ 0*04 88 KEVDPAGHSYIMAGEA8, MAGEA9, MAGEA9B A*01/ B*49 89 EVYDGREHSA MAGEA1 A*01/ Y A*25 90YEDHFPLLF MAGEA10 A*01/ B*18 91 CLVLVIVLLY SLC6A3 A*01 92 TTDDTTAMASAMAGEA10 A*01 S 93 HLKILSPIY UMODL1 A*01 94 KPSAVKDSIY MMP26 A*01/ A*2695 SSDPKAVMF MMP12 A*01/ 0*05 96 TATLLIVRY UMODL1 A*01 97 FPAPPAHWFYCYP4Z1, CYP4Z2P A*01 98 NFSDLVFTY SOX11 A*01/ A*29 99 AADSNPSEL RLN1A*01/ 0*05 100 TTSSAISWILY CYP4Z1 A*01 101 SITDVDFIY DNAH8 A*01 102STIRGELFF MMP11 A*01/ B*57 103 ITDTLIHLM ESR1 A*01/ 0*05 104 ITDTLIHLESR1 A*01/ 0*05 105 VVFDKSDLAKY SLC45A3 A*01/ A*29 106 EVVEGKEWGS NPSR1A*01/ FY A*26 107 TTENSGNYY FCRL5 A*01 108 NSNLKFLEV NLRP7 A*01 109ISEDKSISF LOC100996718, OR9G1, OR9G9 A*01/ B*15 110 IGDKVDAVY MMP13 A*01111 TPIPFDKILY COL10A1 A*01/ B*35 112 KASSVSAEDGY TTC6 A*01/ B*57 113ASCRSSAEY LAMC2 A*01 114 AVAAAAGASLY MSX1 A*01 115 NEIDIHSIYFY HEPHL1A*01/ B*18 116 RSDIGEFEW FAM111B A*01/ B*58 117 SPAKQFNIY FAM111B A*01/B*35 118 LTWAHSAKY LOXL4 A*01 119 TVFDENLSRY HEPHL1 A*01/ 0*12 120LVDENQSWY HEPHL1 A*01 121 SADEAHGLL NFE2L3 A*01/ 0*05 122 ISEAPLTEVSLC45A2 A*01/ 0*08 123 LLKAKDILY ACSM1 A*01 124 FLKVTGYDKDD HMCN1 A*01 Y125 FQYELRELY HMCN1 A*01/ B*15 126 TTDPKKFQY HMCN1 A*01 127 VPFNLITEYSLC45A2 A*01/ B*35 128 YTEFVDATFTK HEPHL1 A*01 129 STIDFRAGF HMCN1 A*01/B*57 130 YIGLKGLYF SLC45A2 A*01/ A*23 131 LEDGIEQSAY G2E3 A*01 132RTHIGYKVY HMCN1 A*01 133 ITDVGPGNY LOXL4 A*01 134 SAPSSSGSPLY T A*01 135TFDKQIVLL F5 A*01/ 0*04 136 RRLNFSGFGY ASCL1 A*01/ B*27 137 EAYLERIGYNAT1, NAT2 A*01/ B*15 138 IPVHDSVGVTY PTPRZ1 A*01/ B*35 139 PVHDSVGVTYPTPRZ1 A*01 140 SQHIFTVSY PTPRZ1 A*01/ B*15 141 DAVAPGREY F5 A*01/ B*35142 IEKFAVLY PTPRZ1 A*01/ B*18 143 HVSGQMLYF PGR A*01/ A*29 144RTIEGDFLW BCAN A*01/ A*32 145 LSDAVHVEF FCRL3 A*01/ 0*05 146 LCATVCGTEQYMMP16 A*01 147 AQVQDTGRY HMCN1 A*01 148 GTKQWVHARY FCER2 A*01/ A*30 149PIMSSSQALY HMCN1 A*01 150 FTTLSDLQTNM MEIOB A*01 A 151 YEVDTKLLSL MEIOBA*01 152 YLEDRPLSQ LOXL4 A*01 153 HSIEVFTHY LOXL4 A*01 154 SIEVFTHYLOXL4 A*01 155 HTMEVTVY PMEL A*01/ B*15 156 STALSILLL BCAN A*01 157GLIEVVTGY IGF1R A*01/ B*15 158 EVTDRNMLAF TRPS1 A*01/ A*25 159RQAPGPARDY KRT13, KRT17 A*01/ B*15 160 EVLGEEMYAY LOC101060117, TTC6A*01 161 EAAPDIMHY GREB1 A*01/ B*35 162 IADNPQLSFY ABCC11 A*01 163KIRAEVLSHY RALGPS2 A*01/ A*30 164 KLAGTVFQY ADAMTS12 A*01 165 VSVYNSYPYNKX3-1 A*01 166 YHRICELLSDY POTEE, POTEF A*01 167 RAVQPGETY F5 A*01/B*15 168 VQPGETYTY F5 A*01/ B*35 169 TVDNANILL JUP, KRT13, KRT17 A*01/0*05 170 VQIAKGMNY EGFR A*01/ B*15 171 ITDFGLAKL EGFR A*01/ 0*05 172FSEPFHLIV FCRLA A*01/ 0*12 173 QSTTGVSHY VCAN A*01 174 TSEVEGLAFVS VCANA*01 Y 175 GLEYEAPKLY DNMT3B A*01 176 HTDLESPSAVY BTBD16 A*01 177LVDGKWQEF BTBD16 A*01 178 TQRTSFQFY ROS1 A*01/ B*15 179 SSTDFTFASWFERMT1 A*01/ A*25 180 AQISDTGRY HMCN1 A*01 181 SVTDLIGGKW HTR7, HTR7P1A*01/ B*57 182 TQPELSSRY ADAMTS12 A*01/ B*15 183 LADTDLGMTF OR51B4 A*01/0*12 184 KTIQEVAGY EGFR A*01/ B*57 185 NSDESADSEPH PRDM15 A*01 KY 186AVSSGLFFY CTLA4 A*01/ A*29 187 TQKSVQVLAY PTPRZ1 A*01/ B*15 188DIPDYLLQY ELL3 A*01/ A*26 189 FRGVFVHRY STAG3 A*01/ 0*07 190 VSSTVHNLYGREB1 A*01/ B*57 191 FTRAFDQLRM TKTL2 A*01/ A*26 192 LAFYYGMY SLC7A11A*01/ B*15 193 SQNGQLIHY SLC5A4 A*01 194 CYTADNEMGY ROS1 A*01 195YTADNEMGYY ROS1 A*01 196 RLAQYTIERY GABRP A*01/ B*15 197 NDEIDKLTGYPDE11A A*01 198 KLTDYINANY PTPRZ1 A*01/ B*15 199 LCAAVLAKYUGT1A3, UGT1A5 A*01 200 SLPEFGLTY FERMT1 A*01/ B*15 201 SLPEFGLTYYFERMT1 A*01/ B*46 202 QTDINGGSLK GPR143 A*01 203 LSQDELSKF NLRP2, PYDC2A*01 204 NVKEAPTEY BTLA A*01/ 0*12 205 RMQEGSEVY BTLA A*01/ B*15 206RVFVAVTLY ABCC4 A*01/ B*15 207 LLEGEDAHLTQ JUP, KRT13, KRT17 A*01 Y 208LLISKAEDY IGF1R A*01/ B*35 209 EADPFLKYL CCNA1 A*01/ B*35 210 LLEADPFLKYCCNA1 A*01 211 YLNEWGSRF CDH3 A*01 212 MMTDLTSVY LOC101060622,  MUC3AA*01/ A*29 213 VSDSTTEITY MUC3A A*01 214 VQDPSLPVY SOX30 A*01 215DTLEAATSLY PLEKHG4B A*01 216 NSMLDPLVY HCAR1 A*01 217 LMDEGAVLTL L1TD1A*01/ 0*17 218 FTAQLQLY ROS1 A*01 219 KTELETALYY GOLGA6L2 A*01 220DVERIKDTY LAMA3 A*01 221 TDVERIKDTY LAMA3 A*01 222 GSPDAVVSY MAGEB6A*01/ B*35 223 NAVDVVPSSF NLRP2 A*01 224 RTDEGDNRVW LY6K A*01/ B*57 225STDPNIVRK TAF7L A*01/ A*11 226 QITPKHNGLY PSG11 A*01/ B*15 227ESAPKEVSRY PDE10A A*01/ A*26 228 KSFDDIAKY SSX7 A*01/ B*15 229 MTDVFIDYABCB11 A*01 230 CVIETFHKY TCHHL1 A*01 231 LLPLLVMAY CXCR3 A*01 232RYLNIVHATQL CXCR3 A*01 Y 233 RINSATGQY CLNK A*01/ A*30 234 YTDLTTIQVLRRC15 A*01 235 SIEIDHTQY MYH4 A*01 236 VLDSLLAQY IGF2BP1 A*01 237AQEAAVFLTLY DNAH8 A*01 238 ETDWGLLKGH CAPN6 A*01 TY 239 SSERGSPIEKYTRPS1 A*01 240 EVLDSLLAQY IGF2BP1 A*01/ A*25 241 SLMVASLTY TNFSF4 A*01/B*15 242 GTNLPTLLW CDKAL1 A*01/ B*57 243 LTSEDTGAY CDKAL1 A*01 244VTKYIAGPY PSG4, PSG5, PSG6, PSG7, A*01 PSG9 245 LSDNAANRY MED12L A*01246 ARLEGEIATY KRT36 A*01 247 SMIRVGTNY RCOR2 A*01/ B*15 248 VTDIDELGKCDK6 A*01 249 GVGFTELEY C9orf57 A*01 250 GYVCNACGLY TRPS1 A*01 251GIEMTYETY SYCP2 A*01 252 DTTSHTYLQY ADCY8 A*01/ A*25 253 YLESHGLAY CPA5A*01 254 FLFNDALLY FGD6 A*01/ A*29 255 WELDSLEY KIF26B A*01 256HAFESNNFIY MET A*01/ B*35 257 KSEMNVNMKY MET A*01 258 RPSSVLTIY SLC44A5A*01/ B*15 259 APDEVVALL KLHL35 A*01 260 KPTEDSANVY DCC A*01 261MTEGSTVNTEY TEX15 A*01 262 NVKHFLNDLY TEX15 A*01 263 DCMDTEGSYM FBN3A*01 264 YRDPVFVSL CYP2W1 A*01/ B*39 265 LSDIDSRYI PRAMEF20, PRAMEF21A*01 266 LTDSFLLRF TTPA A*01/ B*57 267 IVADDTVY ADAM7 A*01 268 AILHHLYFYABCC4 A*01/ A*29 269 LPSPAATIWDY MACC1 A*01/ B*35 270 DLKIDLAAQY DNAH17A*01/ A*25 271 VAEPPVVCSY LOC730110, ZNF492, ZNF98 A*01 272 IPQDECLRYPRAMEF1, PRAMEF13, A*01 PRAMEF14, PRAMEF2 273 CGPNEINHFY OR5M8 A*01 274YADIHGDLL PARD6B A*01/ 0*05 275 ESDEMENLLTY SERHL, SERHL2 A*01 276QITSFASGTSY MED12L A*01 277 LPAPGFLFY GDPGP1 A*01/ B*35 278 AATVKSDIYTEX14 A*01 279 LMTVLLKY SLC16A14 A*01 280 TTEMVSNESVD MET A*01 Y 281YPDLSELLM NUP155 A*01/ B*35 282 QAMPSWPTAA NRG3 A*01 Y 283 ETILVSSSYSLC10A5 A*01 284 TCSHTFVYY VWDE A*01 285 VLPHHSEGACV FRAS1 A*01/ Y B*15286 ATDMEGNLNY CDH4 A*01 287 ENSIEDLQY CCDC83 A*01 288 TEEKFVSY TSPEARA*01 289 YTSHEDIGY SLC16A14 A*01 290 GQFTGTAGAC WT1 A*01/ RY B*15 291TSDVTGSLTY GPR31 A*01 292 VLDFAPPGASA WT1 A*01 Y 293 IISVLIAIY SLC6A5A*01 294 MMEMEGMY DNAH8 A*01 295 GQRLDEAMISY MYO3B A*01/ B*15 296HMLAAMAY OR8G1, OR8G2, OR8G5 A*01 297 RLDEAMISY MYO3B A*01 298 KFDVINHYFOR8G5 A*01 299 EVDSVALSL VRTN A*01 300 VSINPNSGDIY PCDH19 A*01 301ESQTCASDY DCBLD1 A*01 302 FYLSTPENYHY MYO10 A*01/ A*29 303 GFGGLSSQGVNKX6-3 A*01 YY 304 FSENLIYTYI MYO1H A*01 305 YADLLIYTY MYO3B A*01 306KSFETTVRY FMN1 A*01/ 0*12 307 DTDDRELRY FREM2 A*01 308 ELAAGQVVY FREM2A*01/ B*18 309 EVDRNLIQY FREM2 A*01 310 KAFQELGVRY FREM2 A*01/ B*57 311TVTDGTHTDFY FREM2, FREM3 A*01 312 VTDGINPLI FREM2 A*01 313 VTDGTHTDFYFREM2, FREM3 A*01 314 PPEANSLQGAL DBX2 A*01 Y 315 VLKIELETY CCDC6 A*01/B*15 316 YTCEECGQAF ZNF479, ZNF679, ZNF727, A*01 ZNF733P, ZNF734P 317EDLLEVLDMY KCNH7 A*01/ B*18 318 YMTSMALNY ITGAE A*01 319 FTDPHIITF VWDEA*01/ 0*04 320 QALQDKLQTFY VWDE A*01 321 DGIADASNLSYLOC100124692,  LOC93432 A*01 Y 322 FSELNPLALY TDRD5 A*01 323 KTLQKPVLPLYDNMBP A*01/ A*30 324 RTGIFPYRF DNMBP A*01/ B*57 325 LQKPVLPLY DNMBPA*01/ B*15 326 STSRLTLFS DMXL1 A*01 327 IMLSVDQHLY RAPGEF5 A*01 328LLDEDNNIKL HUNK A*01 329 NTDSMTLNNTA EFCAB5 A*01 Y 330 EGELSEGEHW ZNF827A*01 Y 331 YLYQAPGSLAL IRX2 A*01 Y 332 SLISFKYTSY FRAS1 A*01 333LSDPQAELQFY PCDHGB6 A*01 334 PSSMPECLSY SOX30 A*01 335 PSSMPECLSYY SOX30A*01 336 ATNIQLNIDTY CCDC175 A*01 337 FTESNQYNIY FSTL4 A*01 338YSPDSFNVSW USH2A A*01 339 ESMDIFPLGW SFMBT1 A*01/ A*25 340 SVDSNLVAYDMXL1 A*01 341 PANYLGKMTY ARHGEF38 A*01/ B*15 342 QTYMDGLLHY LAMA3 A*01/B*15 343 YFGNYFTYY C5orf34 A*01 344 AVNALQSVY CPA6 A*01/ B*15 345NTMDAVPRIDH SLC12A2 A*01 Y 346 VAGLEAGVLY UMODL1 A*01 347 SADHPGLTFFREM2 A*01/ 0*05 348 DSTDGCLLSF TBC1D16 A*01 349 HLLSVSLYYOR5AC2, OR5H14, OR5H2, OR5H6 A*01 350 LTDPQVSYV FARP2 A*01 351 VLDPMLDFYFAM46D A*01 352 YPVVVAESMY FAM46D A*01 353 RLNGSVASY FAM46D A*01 354EIIRYIFAY HPGDS A*01/ A*26 355 MADRGEARL RMI2 A*01/ 0*04 356 NSENHILKYC12orf56 A*01 357 MSPDIALLYL OVCH1 A*01 358 YMSPDIALLY OVCH1 A*01 359NKEINYFMY ADCY10 A*01/ A*32 360 RFDDINQEF DNAH17 A*01/ 0*04 361FTAEEGQLY AR A*01 362 SGALDEAAAY AR A*01 363 LTDRDVSFY MROH2B A*01 364DTGYLQLYY STON2 A*01 365 FVDTKVPEH ABCC12 A*01 366 ITVDVRDEF STON2 A*01/B*57 367 LTDTGYLQLY STON2 A*01 368 ESAATGQLDY TNR A*01 369 AVMEAAFVYTXNDC16 A*01/ A*29 370 RLSTIRHLY MXRA5 A*01/ A*32 371 WSDSTSQTIYSH3PXD2A A*01 372 SRSDFEWVY DEGS2 A*01/ 0*07 373 FHADSDDESF CDCA7 A*01/B*38 374 LTSVVVTLW CDK6 A*01/ B*57 375 ASSLDSLHY PTPRT A*01 376EDDEDEDLY HSD17B4 A*01 377 YADPSANRDLL ST8SIA5 A*01/ B*35 378 TAKAPSTEYGALNT5 A*01/ B*15 379 SLIIDDTEY FSIP2 A*01/ B*35 380 VACGNNPVY FSIP2A*01/ B*15 381 ETSFSTSHY NRG1 A*01/ A*26 382 YEPATMEQY MYO10 A*01 383PPDHAVGRTKY GCNT2 A*01 384 RFRSITQSYY RAB30 A*01/ A*30 385 SANALILTYRAB30 A*01/ B*35

TABLE 2 Additional peptides according to the presentinvention with no prior known cancer association. SEQ ID No. SequenceGene(s) HLA allotype 386 NSALNPLLY KISS1R A*01 387 LMEKEDYHSLY TYR, TYRLA*01 388 YTAHVGYSMY MSX1 A*01 389 YYDLVESTF DNTT A*01 390 FSEPFHLIVSYFCRLA A*01 391 GSNPARYEF MAGEA4 A*01/ B*57 392 TQHFVQENY MAGEA3A*01/ B*15 393 QVWGGQPVY PMEL A*01/ B*35 394 QVPLDCVLY PMEL A*01 395ILKGGSGTY PMEL A*01/ B*15 396 LPDPNVQKY PRDM15 A*01/ C*04 397 NSAINPLIYNPSR1 A*01 398 YYYDTHTNTY SLC12A2 A*01/ C*14

TABLE 3 Peptides useful for e.g. personalized cancer therapies. SEQ IDNo. Sequence Gene(s) HLA allotype 399 YVGKEHMFY MAGEA9, MAGEA9B A*01 400NTDNNLAVY CXorf61 A*01 401 VWSNVTPLKF MMP12 A*01 402 VLYPVPLESY PRAMEA*01 403 SADDIRGIQSLY MMP12 A*01 404 FVDNQYWRY MMP12 A*01 405 STDIGALMYMMP1 A*01 406 SDVTPLTF MMP11 A*01 407 VSIRNTLLY FLT3 A*01 408 VWSDVTPLTFMMP11 A*01 409 YTFRYPLSL MMP11 A*01 410 KTWAHCMSY KISS1R A*01 411LLDAEPPILY ESR1 A*01 412 AAAANAQVY ESR1 A*01 413 TDTLIHLM ESR1 A*01 414LTEGHSGNYY FCRL5 A*01 415 VWSDVTPLNF MMP13 A*01 416 LSSPVHLDF FCRL5 A*01417 KLDRSVFTAY FAM111B A*01 418 LLDEGAMLLY NLRP7 A*01 419 FPTEVTPHAFPTPRZ1 A*01 420 VTDLEMPHY PTPRZ1 A*01 421 RSDPGGGGLA MEX3B A*01 Y 422ALNPYQYQY DLX5 A*01 423 LLDEGAKLLY NLRP2 A*01 424 NVDPVQHTY AGRN A*01425 VTEEPQRLFY BMP A*01 426 RSFNGLLTMY LAMB3 A*01 427 LTDYINANY PTPRZ1A*01 428 IINESLLFY GPR143 A*01 429 VSDSECLSRY LAMA1 A*01 430 NTDPTAPPYCDH3 A*01 431 ASSTDSASYY APOB A*01 432 LQSSGLTLLL MSLNL A*01

As can be seen from the above tables, many of the preferred peptides ofthe invention are surprisingly able to bind to at last two HLAallotypes. This allows for, in particular, broader applicability ofthese peptides.

The present invention furthermore generally relates to the peptidesaccording to the present invention for use in the treatment ofproliferative diseases, such as, for example, acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer.

Particularly preferred are the peptides—alone or incombination—according to the present invention selected from the groupconsisting of SEQ ID NO: 1 to SEQ ID NO: 398. More preferred are thepeptides—alone or in combination—selected from the group consisting ofSEQ ID NO: 1 to SEQ ID NO: 21 (see Table), and their uses in theimmunotherapy of acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer, and preferably acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer.

Thus, another aspect of the present invention relates to the use of thepeptides according to the present invention for the—preferablycombined—treatment of a proliferative disease selected from the group ofacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

The present invention furthermore relates to peptides according to thepresent invention that have the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or—in an elongatedform, such as a length-variant—MHC class-II.

The present invention further relates to the peptides according to thepresent invention wherein said peptides (each) consist or consistessentially of an amino acid sequence according to SEQ ID NO: 1 to SEQID NO: 398.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to thepresent invention, wherein said peptide is part of a fusion protein, inparticular fused to the N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii) or fused to (or into thesequence of) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the present invention. The present inventionfurther relates to the nucleic acid according to the present inventionthat is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing and/or expressing a nucleic acid according to the presentinvention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use in thetreatment of diseases and in medicine, in particular in the treatment ofcancer.

The present invention further relates to antibodies that are specificagainst the peptides according to the present invention or complexes ofsaid peptides according to the present invention with MHC, and methodsof making these.

The present invention further relates to T-cell receptors (TCRs), inparticular soluble TCR (sTCRs) and cloned TCRs engineered intoautologous or allogeneic T cells, and methods of making these, as wellas NK cells or other cells bearing said TCR or cross-reacting with saidTCRs.

The antibodies and TCRs are additional embodiments of theimmunotherapeutic use of the peptides according to the invention athand.

The present invention further relates to a host cell comprising anucleic acid according to the present invention or an expression vectoras described before. The present invention further relates to the hostcell according to the present invention that is an antigen presentingcell, and preferably is a dendritic cell.

The present invention further relates to a method for producing apeptide according to the present invention, said method comprisingculturing the host cell according to the present invention, andisolating the peptide from said host cell or its culture medium.

The present invention further relates to said method according to thepresent invention, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell.

The present invention further relates to the method according to thepresent invention, wherein the antigen-presenting cell comprises anexpression vector capable of expressing or expressing said peptidecontaining SEQ ID No. 1 to SEQ ID No.: 398, preferably containing SEQ IDNo. 1 to SEQ ID No. 21, or a variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellselectively recognizes a cell which expresses a polypeptide comprisingan amino acid sequence according to the present invention.

The present invention further relates to a method of killing targetcells in a patient which target cells aberrantly express a polypeptidecomprising any amino acid sequence according to the present invention,the method comprising administering to the patient an effective numberof T cells as produced according to the present invention.

The present invention further relates to the use of any peptide asdescribed, the nucleic acid according to the present invention, theexpression vector according to the present invention, the cell accordingto the present invention, the activated T lymphocyte, the T cellreceptor or the antibody or other peptide- and/or peptide-MHC-bindingmolecules according to the present invention as a medicament or in themanufacture of a medicament. Preferably, said medicament is activeagainst cancer.

Preferably, said medicament is a cellular therapy, a vaccine or aprotein based on a soluble TCR or antibody.

The present invention further relates to a use according to the presentinvention, wherein said cancer cells are acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer, and preferably acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancercells.

The present invention further relates to biomarkers based on thepeptides according to the present invention, herein called “targets”that can be used in the diagnosis of cancer, preferably acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.The marker can be over-presentation of the peptide(s) themselves, orover-expression of the corresponding gene(s). The markers may also beused to predict the probability of success of a treatment, preferably animmunotherapy, and most preferred an immunotherapy targeting the sametarget that is identified by the biomarker. For example, an antibody orsoluble TCR can be used to stain sections of the tumor to detect thepresence of a peptide of interest in complex with MHC.

Optionally the antibody carries a further effector function such as animmune stimulating domain or toxin.

The present invention also relates to the use of these novel targets inthe context of cancer treatment.

Stimulation of an immune response is dependent upon the presence ofantigens recognized as foreign by the host immune system. The discoveryof the existence of tumor associated antigens has raised the possibilityof using a host's immune system to intervene in tumor growth. Variousmechanisms of harnessing both the humoral and cellular arms of theimmune system are currently being explored for cancer immunotherapy.

Specific elements of the cellular immune response are capable ofspecifically recognizing and destroying tumor cells. The isolation ofT-cells from tumor-infiltrating cell populations or from peripheralblood suggests that such cells play an important role in natural immunedefense against cancer. CD8-positive T-cells in particular, whichrecognize class I molecules of the major histocompatibility complex(MHC)-bearing peptides of usually 8 to 10 amino acid residues derivedfrom proteins or defect ribosomal products (DRIPS) located in thecytosol, play an important role in this response. The MHC-molecules ofthe human are also designated as human leukocyte-antigens (HLA).

As used herein and except as noted otherwise all terms are defined asgiven below.

The term “T-cell response” means the specific proliferation andactivation of effector functions induced by a peptide in vitro or invivo. For MHC class I restricted cytotoxic T cells, effector functionsmay be lysis of peptide-pulsed, peptide-precursor pulsed or naturallypeptide-presenting target cells, secretion of cytokines, preferablyInterferon-gamma, TNF-alpha, or IL-2 induced by peptide, secretion ofeffector molecules, preferably granzymes or perforins induced bypeptide, or degranulation.

The term “peptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thepeptides are preferably 9 amino acids in length but can be as short as 8amino acids in length, and as long as 10, 11, 12, 13, or 14 or longer,and in case of MHC class II peptides (elongated variants of the peptidesof the invention) they can be as long as 15, 16, 17, 18, 19 or 20 ormore amino acids in length.

Furthermore, the term “peptide” shall include salts of a series of aminoacid residues, connected one to the other typically by peptide bondsbetween the alpha-amino and carbonyl groups of the adjacent amino acids.Preferably, the salts are pharmaceutical acceptable salts of thepeptides, such as, for example, the chloride or acetate(trifluoroacetate) salts. It has to be noted that the salts of thepeptides according to the present invention differ substantially fromthe peptides in their state(s) in vivo, as the peptides are not salts invivo.

The term “peptide” shall also include “oligopeptide”. The term“oligopeptide” is used herein to designate a series of amino acidresidues, connected one to the other typically by peptide bonds betweenthe alpha-amino and carbonyl groups of the adjacent amino acids. Thelength of the oligopeptide is not critical to the invention, as long asthe correct epitope or epitopes are maintained therein. Theoligopeptides are typically less than about 30 amino acid residues inlength, and greater than about 15 amino acids in length.

The term “polypeptide” designates a series of amino acid residues,connected one to the other typically by peptide bonds between thealpha-amino and carbonyl groups of the adjacent amino acids. The lengthof the polypeptide is not critical to the invention as long as thecorrect epitopes are maintained. In contrast to the terms peptide oroligopeptide, the term polypeptide is meant to refer to moleculescontaining more than about 30 amino acid residues.

A peptide, oligopeptide, protein or polynucleotide coding for such amolecule is “immunogenic” (and thus is an “immunogen” within the presentinvention), if it is capable of inducing an immune response. In the caseof the present invention, immunogenicity is more specifically defined asthe ability to induce a T-cell response. Thus, an “immunogen” would be amolecule that is capable of inducing an immune response, and in the caseof the present invention, a molecule capable of inducing a T-cellresponse. In another aspect, the immunogen can be the peptide, thecomplex of the peptide with MHC, oligopeptide, and/or protein that isused to raise specific antibodies or TCRs against it.

A class I T cell “epitope” requires a short peptide that is bound to aclass I MHC receptor, forming a ternary complex (MHC class I alphachain, beta-2-microglobulin, and peptide) that can be recognized by a Tcell bearing a matching T-cell receptor binding to the MHC/peptidecomplex with appropriate affinity. Peptides binding to MHC class Imolecules are typically 8-14 amino acids in length, and most typically 9amino acids in length.

In humans there are three different genetic loci that encode MHC class Imolecules (the MHC-molecules of the human are also designated humanleukocyte antigens (HLA)): HLA-A, HLA-B, and HLA-C. HLA-A*01, HLA-A*02,and HLA-B*07 are examples of different MHC class I alleles that can beexpressed from these loci.

TABLE 4 Expression frequencies F of HLA-A*02, HLA-A*01, HLA-A*03,HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 serotypes. Haplotypefrequencies Gf are derived from a study which used HLA-typing data froma registry of more than 6.5 million volunteer donors in the U.S.(Gragert et al., 2013). The haplotype frequency is the frequency of adistinct allele on an individual chromosome. Due to the diploid set ofchromosomes within mammalian cells, the frequency of genotypicoccurrence of this allele is higher and can be calculated employing theHardy-Weinberg principle (F = 1 − (1-Gf)²). Calculated phenotype fromallele Allele Population frequency (F) A*02 African (N = 28557) 32.3%European Caucasian (N = 1242890) 49.3% Japanese (N = 24582) 42.7%Hispanic, S + Cent Amer. 46.1% (N = 146714) Southeast Asian (N = 27978)30.4% A*01 African (N = 28557) 10.2% European Caucasian (N = 1242890)30.2% Japanese (N = 24582)  1.8% Hispanic, S + Cent Amer. 14.0% (N =146714) Southeast Asian (N = 27978) 21.0% A*03 African (N = 28557) 14.8%European Caucasian (N = 1242890) 26.4% Japanese (N = 24582)  1.8%Hispanic, S + Cent Amer. 14.4% (N = 146714) Southeast Asian (N = 27978)10.6% A*24 African (N = 28557)  2.0% European Caucasian (N = 1242890) 8.6% Japanese (N = 24582) 35.5% Hispanic, S + Cent Amer. 13.6% (N =146714) Southeast Asian (N = 27978) 16.9% B*07 African (N = 28557) 14.7%European Caucasian (N = 1242890) 25.0% Japanese (N = 24582) 11.4%Hispanic, S + Cent Amer. 12.2% (N = 146714) Southeast Asian (N = 27978)10.4% B*08 African (N = 28557)  6.0% European Caucasian (N = 1242890)21.6% Japanese (N = 24582)  1.0% Hispanic, S + Cent Amer.  7.6% (N =146714) Southeast Asian (N = 27978)  6.2% B*44 African (N = 28557) 10.6%European Caucasian (N = 1242890) 26.9% Japanese (N = 24582) 13.0%Hispanic, S + Cent Amer. 18.2% (N = 146714) Southeast Asian (N = 27978)13.1%

The peptides of the invention, preferably when included into a vaccineof the invention as described herein bind to A*01. A vaccine may alsoinclude pan-binding MHC class II peptides. Therefore, the vaccine of theinvention can be used to treat cancer in patients that areA*01-positive, whereas no selection for MHC class II allotypes isnecessary due to the pan-binding nature of these peptides.

If A*01 peptides of the invention are combined with peptides binding toanother allele, for example A*24, a higher percentage of any patientpopulation can be treated compared with addressing either MHC class Iallele alone. While in most populations less than 50% of patients couldbe addressed by either allele alone, a vaccine comprising HLA-A*24 andHLA-A*02 epitopes can treat at least 60% of patients in any relevantpopulation. Specifically, the following percentages of patients will bepositive for at least one of these alleles in various regions: USA 61%,Western Europe 62%, China 75%, South Korea 77%, Japan 86% (calculatedfrom www.allelefrequencies.net).

TABLE 5 HLA alleles coverage in European Caucasian population(calculated from (Gragert et al., 2013)). coverage (at least combinedone A- combined combined with B*07 allele) with B*07 with B*44 and B*44A*02/A*01 70% 78% 78% 84% A*02/A*03 68% 76% 76% 83% A*02/A*24 61% 71%71% 80% A*′01/A*03 52% 64% 65% 75% A*01/A*24 44% 58% 59% 71% A*03/A*2440% 55% 56% 69% A*02/A*01/A*03 84% 88% 88% 91% A*02/A*01/A*24 79% 84%84% 89% A*02/A*03/A*24 77% 82% 83% 88% A*01/A*03/A*24 63% 72% 73% 81%A*02/A*01/A*03/A*24 90% 92% 93% 95%

In a preferred embodiment, the term “nucleotide sequence” refers to aheteropolymer of deoxyribonucleotides.

The nucleotide sequence coding for a particular peptide, oligopeptide,or polypeptide may be naturally occurring or they may be syntheticallyconstructed. Generally, DNA segments encoding the peptides,polypeptides, and proteins of this invention are assembled from cDNAfragments and short oligonucleotide linkers, or from a series ofoligonucleotides, to provide a synthetic gene that is capable of beingexpressed in a recombinant transcriptional unit comprising regulatoryelements derived from a microbial or viral operon.

As used herein the term “a nucleotide coding for (or encoding) apeptide” refers to a nucleotide sequence coding for the peptideincluding artificial (man-made) start and stop codons compatible for thebiological system the sequence is to be expressed by, for example, adendritic cell or another cell system useful for the production of TCRs.

As used herein, reference to a nucleic acid sequence includes bothsingle stranded and double stranded nucleic acid. Thus, for example forDNA, the specific sequence, unless the context indicates otherwise,refers to the single strand DNA of such sequence, the duplex of suchsequence with its complement (double stranded DNA) and the complement ofsuch sequence.

The term “coding region” refers to that portion of a gene which eithernaturally or normally codes for the expression product of that gene inits natural genomic environment, i.e., the region coding in vivo for thenative expression product of the gene.

The coding region can be derived from a non-mutated (“normal”), mutatedor altered gene, or can even be derived from a DNA sequence, or gene,wholly synthesized in the laboratory using methods well known to thoseof skill in the art of DNA synthesis.

The term “expression product” means the polypeptide or protein that isthe natural translation product of the gene and any nucleic acidsequence coding equivalents resulting from genetic code degeneracy andthus coding for the same amino acid(s).

The term “fragment”, when referring to a coding sequence, means aportion of DNA comprising less than the complete coding region, whoseexpression product retains essentially the same biological function oractivity as the expression product of the complete coding region.

The term “DNA segment” refers to a DNA polymer, in the form of aseparate fragment or as a component of a larger DNA construct, which hasbeen derived from DNA isolated at least once in substantially pure form,i.e., free of contaminating endogenous materials and in a quantity orconcentration enabling identification, manipulation, and recovery of thesegment and its component nucleotide sequences by standard biochemicalmethods, for example, by using a cloning vector. Such segments areprovided in the form of an open reading frame uninterrupted by internalnon-translated sequences, or introns, which are typically present ineukaryotic genes. Sequences of non-translated DNA may be presentdownstream from the open reading frame, where the same do not interferewith manipulation or expression of the coding regions.

The term “primer” means a short nucleic acid sequence that can be pairedwith one strand of DNA and provides a free 3′-OH end at which a DNApolymerase starts synthesis of a deoxyribonucleotide chain.

The term “promoter” means a region of DNA involved in binding of RNApolymerase to initiate transcription.

The term “isolated” means that the material is removed from its originalenvironment (e.g., the natural environment, if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition, and still be isolated inthat such vector or composition is not part of its natural environment.

The polynucleotides, and recombinant or immunogenic polypeptides,disclosed in accordance with the present invention may also be in“purified” form. The term “purified” does not require absolute purity;rather, it is intended as a relative definition, and can includepreparations that are highly purified or preparations that are onlypartially purified, as those terms are understood by those of skill inthe relevant art. For example, individual clones isolated from a cDNAlibrary have been conventionally purified to electrophoretichomogeneity. Purification of starting material or natural material to atleast one order of magnitude, preferably two or three orders, and morepreferably four or five orders of magnitude is expressly contemplated.Furthermore, a claimed polypeptide which has a purity of preferably99.999%, or at least 99.99% or 99.9%; and even desirably 99% by weightor greater is expressly encompassed.

The nucleic acids and polypeptide expression products disclosedaccording to the present invention, as well as expression vectorscontaining such nucleic acids and/or such polypeptides, may be in“enriched form”. As used herein, the term “enriched” means that theconcentration of the material is at least about 2, 5, 10, 100, or 1000times its natural concentration (for example), advantageously 0.01%, byweight, preferably at least about 0.1% by weight. Enriched preparationsof about 0.5%, 1%, 5%, 10%, and 20% by weight are also contemplated. Thesequences, constructs, vectors, clones, and other materials comprisingthe present invention can advantageously be in enriched or isolatedform. The term “active fragment” means a fragment, usually of a peptide,polypeptide or nucleic acid sequence, that generates an immune response(i.e., has immunogenic activity) when administered, alone or optionallywith a suitable adjuvant or in a vector, to an animal, such as a mammal,for example, a rabbit or a mouse, and also including a human, suchimmune response taking the form of stimulating a T-cell response withinthe recipient animal, such as a human. Alternatively, the “activefragment” may also be used to induce a T-cell response in vitro.

As used herein, the terms “portion”, “segment” and “fragment”, when usedin relation to polypeptides, refer to a continuous sequence of residues,such as amino acid residues, which sequence forms a subset of a largersequence. For example, if a polypeptide were subjected to treatment withany of the common endopeptidases, such as trypsin or chymotrypsin, theoligopeptides resulting from such treatment would represent portions,segments or fragments of the starting polypeptide. When used in relationto polynucleotides, these terms refer to the products produced bytreatment of said polynucleotides with any of the endonucleases.

In accordance with the present invention, the term “percent identity” or“percent identical”, when referring to a sequence, means that a sequenceis compared to a claimed or described sequence after alignment of thesequence to be compared (the “Compared Sequence”) with the described orclaimed sequence (the “Reference Sequence”). The percent identity isthen determined according to the following formula:

percent identity=100[1−(C/R)]

wherein C is the number of differences between the Reference Sequenceand the Compared Sequence over the length of alignment between theReference Sequence and the Compared Sequence, wherein(i) each base or amino acid in the Reference Sequence that does not havea corresponding aligned base or amino acid in the Compared Sequence and(ii) each gap in the Reference Sequence and(iii) each aligned base or amino acid in the Reference Sequence that isdifferent from an aligned base or amino acid in the Compared Sequence,constitutes a difference and(iiii) the alignment has to start at position 1 of the alignedsequences; and R is the number of bases or amino acids in the ReferenceSequence over the length of the alignment with the Compared Sequencewith any gap created in the Reference Sequence also being counted as abase or amino acid.

If an alignment exists between the Compared Sequence and the ReferenceSequence for which the percent identity as calculated above is aboutequal to or greater than a specified minimum Percent Identity then theCompared Sequence has the specified minimum percent identity to theReference Sequence even though alignments may exist in which the hereinabove calculated percent identity is less than the specified percentidentity.

As mentioned above, the present invention thus provides a peptidecomprising a sequence that is selected from the group of consisting ofSEQ ID NO: 1 to SEQ ID NO: 398 or a variant thereof which is 88%homologous to SEQ ID NO: 1 to SEQ ID NO: 398, or a variant thereof thatwill induce T cells cross-reacting with said peptide. The peptides ofthe invention have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or elongated versions of saidpeptides to class II.

In the present invention, the term “homologous” refers to the degree ofidentity (see percent identity above) between sequences of two aminoacid sequences, i.e. peptide or polypeptide sequences. Theaforementioned “homology” is determined by comparing two sequencesaligned under optimal conditions over the sequences to be compared. Sucha sequence homology can be calculated by creating an alignment using,for example, the ClustalW algorithm. Commonly available sequenceanalysis software, more specifically, Vector NTI, GENETYX or other toolsare provided by public databases.

A person skilled in the art will be able to assess, whether T cellsinduced by a variant of a specific peptide will be able to cross-reactwith the peptide itself (Appay et al., 2006; Colombetti et al., 2006;Fong et al., 2001; Zaremba et al., 1997).

By a “variant” of the given amino acid sequence the inventors mean thatthe side chains of, for example, one or two of the amino acid residuesare altered (for example by replacing them with the side chain ofanother naturally occurring amino acid residue or some other side chain)such that the peptide is still able to bind to an HLA molecule insubstantially the same way as a peptide consisting of the given aminoacid sequence in consisting of SEQ ID NO: 1 to SEQ ID NO: 398. Forexample, a peptide may be modified so that it at least maintains, if notimproves, the ability to interact with and bind to the binding groove ofa suitable MHC molecule, such as HLA-A*02 or -DR, and in that way, it atleast maintains, if not improves, the ability to bind to the TCR ofactivated T cells.

These T cells can subsequently cross-react with cells and kill cellsthat express a polypeptide that contains the natural amino acid sequenceof the cognate peptide as defined in the aspects of the invention. Ascan be derived from the scientific literature and databases (Godkin etal., 1997; Rammensee et al., 1999), certain positions of HLA bindingpeptides are typically anchor residues forming a core sequence fittingto the binding motif of the HLA receptor, which is defined by polar,electrophysical, hydrophobic and spatial properties of the polypeptidechains constituting the binding groove. Thus, one skilled in the artwould be able to modify the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO 398, by maintaining the known anchor residues, and wouldbe able to determine whether such variants maintain the ability to bindMHC class I or II molecules. The variants of the present inventionretain the ability to bind to the TCR of activated T cells, which cansubsequently cross-react with and kill cells that express a polypeptidecontaining the natural amino acid sequence of the cognate peptide asdefined in the aspects of the invention.

The original (unmodified) peptides as disclosed herein can be modifiedby the substitution of one or more residues at different, possiblyselective, sites within the peptide chain, if not otherwise stated.Preferably those substitutions are located at the end of the amino acidchain. Such substitutions may be of a conservative nature, for example,where one amino acid is replaced by an amino acid of similar structureand characteristics, such as where a hydrophobic amino acid is replacedby another hydrophobic amino acid. Even more conservative would bereplacement of amino acids of the same or similar size and chemicalnature, such as where leucine is replaced by isoleucine. In studies ofsequence variations in families of naturally occurring homologousproteins, certain amino acid substitutions are more often tolerated thanothers, and these are often show correlation with similarities in size,charge, polarity, and hydrophobicity between the original amino acid andits replacement, and such is the basis for defining “conservativesubstitutions.”

Conservative substitutions are herein defined as exchanges within one ofthe following five groups: Group 1-small aliphatic, nonpolar or slightlypolar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negativelycharged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar,positively charged residues (His, Arg, Lys); Group 4-large, aliphatic,nonpolar residues (Met, Leu, Ile, Val, Cys); and Group 5-large, aromaticresidues (Phe, Tyr, Trp).

In an aspect, conservative substitutions may include those, which aredescribed by Dayhoff in “The Atlas of Protein Sequence and Structure.Vol. 5”, Natl. Biomedical Research, the contents of which areincorporated by reference in their entirety. For example, in an aspect,amino acids, which belong to one of the following groups, can beexchanged for one another, thus, constituting a conservative exchange:Group 1: alanine (A), proline (P), glycine (G), asparagine (N), serine(S), threonine (T); Group 2: cysteine (C), serine (S), tyrosine (Y),threonine (T); Group 3: valine (V), isoleucine (I), leucine (L),methionine (M), alanine (A), phenylalanine (F); Group 4: lysine (K),arginine (R), histidine (H); Group 5: phenylalanine (F), tyrosine (Y),tryptophan (W), histidine (H); and Group 6: aspartic acid (D), glutamicacid (E). In an aspect, a conservative amino acid substitution may beselected from the following of T→A, G→A, A→d, T→V, A→M, T→I, A→V, T→G,and/or T→S.

In an aspect, a conservative amino acid substitution may include thesubstitution of an amino acid by another amino acid of the same class,for example, (1) nonpolar: Ala, Val, Leu, Ile, Pro, Met, Phe, Trp; (2)uncharged polar: Gly, Ser, Thr, Cys, Tyr, Asn, Gln; (3) acidic: Asp,Glu; and (4) basic: Lys, Arg, His. Other conservative amino acidsubstitutions may also be made as follows: (1) aromatic: Phe, Tyr, His;(2) proton donor: Asn, Gln, Lys, Arg, His, Trp; and (3) proton acceptor:Glu, Asp, Thr, Ser, Tyr, Asn, Gln (see, for example, U.S. Pat. No.10,106,805, the contents of which are incorporated by reference in theirentirety).

In another aspect, conservative substitutions may be made in accordancewith Table A. Methods for predicting tolerance to protein modificationmay be found in, for example, Guo et al., Proc. Natl. Acad. Sci., USA,101(25):9205-9210 (2004), the contents of which are incorporated byreference in their entirety.

TABLE A Conservative Amino Acid Substitutions Amino Acid Substitutions(others are known in the art) Ala Ser, Gly, Cys Arg Lys, Gln, His AsnGln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln Asn, Lys,Glu, Asp, Arg Glu Asp, Asn, Gln Gly Pro, Ala, Ser His Asn, Gln, Lys IleLeu, Val, Met, Ala Leu Ile, Val, Met, Ala Lys Arg, Gln, His Met Leu,Ile, Val, Ala, Phe Phe Met, Leu, Tyr, Trp, His Ser Thr, Cys, Ala ThrSer, Val, Ala Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met, Ala, Thr

In another aspect, conservative substitutions may be those shown inTable B under the heading of “conservative substitutions.” If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table B,may be introduced and the products screened if needed.

TABLE B Amino Acid Substitutions Original Residue (naturally occurringamino Conservative acid) Substitutions Exemplary Substitutions Ala (A)Val Val; Leu; Ile Arg (R) Lys Lys; Gln; Asn Asn (N) Gln Gln; His; Asp,Lys; Arg Asp (D) Glu Glu; Asn Cys (C) Ser Ser; Ala Gln (Q) Asn Asn; GluGlu (E) Asp Asp; Gln Gly (G) Ala Ala His (H) Ala Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu (L) Ile Norleucine; Ile;Val; Met; Ala; Phe Lys (K) Arg Arg; Gln; Asn Met (M) Leu Leu; Phe; IlePhe (F) Tyr Leu; Val; Ile; Ala; Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr(T) Ser Ser Trp (W) Tyr Tyr; Phe Tyr (Y) Phe Trp; Phe; Thr; Ser Val (V)Leu Ile; Leu; Met; Phe; Ala; Norleucine

Less conservative substitutions might involve the replacement of oneamino acid by another that has similar characteristics but is somewhatdifferent in size, such as replacement of an alanine by an isoleucineresidue. Highly non-conservative replacements might involve substitutingan acidic amino acid for one that is polar, or even for one that isbasic in character. Such “radical” substitutions cannot, however, bedismissed as potentially ineffective since chemical effects are nottotally predictable and radical substitutions might well give rise toserendipitous effects not otherwise predictable from simple chemicalprinciples.

Of course, such substitutions may involve structures other than thecommon L-amino acids. Thus, D-amino acids might be substituted for theL-amino acids commonly found in the antigenic peptides of the inventionand yet still be encompassed by the disclosure herein. In addition,non-standard amino acids (i.e., other than the common naturallyoccurring proteinogenic amino acids) may also be used for substitutionpurposes to produce immunogens and immunogenic polypeptides according tothe present invention.

If substitutions at more than one position are found to result in apeptide with substantially equivalent or greater antigenic activity asdefined below, then combinations of those substitutions will be testedto determine if the combined substitutions result in additive orsynergistic effects on the antigenicity of the peptide. At most, no morethan 4 positions within the peptide would be simultaneously substituted.

A peptide consisting essentially of the amino acid sequence as indicatedherein can have one or two non-anchor amino acids (see below regardingthe anchor motif) exchanged without that the ability to bind to amolecule of the human major histocompatibility complex (MHC) class-I or-II is substantially changed or is negatively affected, when compared tothe non-modified peptide. In another embodiment, in a peptide consistingessentially of the amino acid sequence as indicated herein, one or twoamino acids can be exchanged with their conservative exchange partners(see herein below) without that the ability to bind to a molecule of thehuman major histocompatibility complex (MHC) class-I or -II issubstantially changed, or is negatively affected, when compared to thenon-modified peptide.

The amino acid residues that do not substantially contribute tointeractions with the T-cell receptor can be modified by replacementwith other amino acid whose incorporation does not substantially affectT-cell reactivity and does not eliminate binding to the relevant MHC.Thus, apart from the proviso given, the peptide of the invention may beany peptide (by which term the inventors include oligopeptide orpolypeptide), which includes the amino acid sequences or a portion orvariant thereof as given.

TABLE 6 Variants and motif of the peptidesaccording to SEQ ID NO: 1, 4, 9, 10, and 12. Position 1 2 3 4 5 6 7 8 9Seq ID No 1 T L D S T R T L Y Variant S S A S E S E A T T A T E T E APosition 1 2 3 4 5 6 7 8 9 Seq ID No 4 R I E A I R A E Y Variant S D S DA S S A T D T D A T T A Position 1 2 3 4 5 6 7 8 9 Seq ID No 9 Q L D S TL D S Y Variant S S A S E S E A T T A T E T E A Position 1 2 3 4 5 6 7 89 Seq ID No 10 V S E R T G I S Y Variant D D A A T D T D A T T APosition 1 2 3 4 5 6 7 8 9 10 Seq ID No 12 Y T D F V G E G L Y Variant SS A S E S E A A E E A

Longer (elongated) peptides may also be suitable. It is possible thatMHC class I epitopes, although usually between 8 and 11 amino acidslong, are generated by peptide processing from longer peptides orproteins that include the actual epitope. It is preferred that theresidues that flank the actual epitope are residues that do notsubstantially affect proteolytic cleavage necessary to expose the actualepitope during processing.

The peptides of the invention can be elongated by up to four aminoacids, that is 1, 2, 3 or 4 amino acids can be added to either end inany combination between 4:0 and 0:4. Combinations of the elongationsaccording to the invention can be found in Table 7.

TABLE 7 Combinations of the elongations of peptides of the inventionC-terminus N-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0 or 1 or 2 or 3 0 0or 1 or 2 or 3 or 4 N-terminus C-terminus 4 0 3 0 or 1 2 0 or 1 or 2 1 0or 1 or 2 or 3 0 0 or 1 or 2 or 3 or 4

The amino acids for the elongation/extension can be the peptides of theoriginal sequence of the protein or any other amino acid(s). Theelongation can be used to enhance the stability or solubility of thepeptides.

Thus, the epitopes of the present invention may be identical tonaturally occurring tumor-associated or tumor-specific epitopes or mayinclude epitopes that differ by no more than four residues from thereference peptide, as long as they have substantially identicalantigenic activity.

In an alternative embodiment, the peptide is elongated on either or bothsides by more than 4 amino acids, preferably to a total length of up to30 amino acids. This may lead to MHC class II binding peptides. Bindingto MHC class II can be tested by methods known in the art.

Accordingly, the present invention provides peptides and variants of MHCclass I epitopes, wherein the peptide or variant has an overall lengthof between 8 and 100, preferably between 8 and 30, and most preferredbetween 8 and 14, namely 8, 9, 10, 11, 12, 13, 14 amino acids, in caseof the elongated class II binding peptides the length can also be 15,16, 17, 18, 19, 20, 21 or 22 amino acids.

Of course, the peptide or variant according to the present inventionwill have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class I or II. Binding of a peptide ora variant to an MHC complex may be tested by methods known in the art.

Preferably, when the T cells specific for a peptide according to thepresent invention are tested against the substituted peptides, thepeptide concentration at which the substituted peptides achieve half themaximal increase in lysis relative to background is no more than about 1mM, preferably no more than about 1 μM, more preferably no more thanabout 1 nM, and still more preferably no more than about 100 pM, andmost preferably no more than about 10 pM. It is also preferred that thesubstituted peptide be recognized by T cells from more than oneindividual, at least two, and more preferably three individuals.

In a particularly preferred embodiment of the invention the peptideconsists or consists essentially of an amino acid sequence according toSEQ ID NO: 1 to SEQ ID NO: 398.

“Consisting essentially of” shall mean that a peptide according to thepresent invention, in addition to the sequence according to any of SEQID NO: 1 to SEQ ID NO 398 or a variant thereof contains additional N-and/or C-terminally located stretches of amino acids that are notnecessarily forming part of the peptide that functions as an epitope forMHC molecules epitope.

Nevertheless, these stretches can be important to provide an efficientintroduction of the peptide according to the present invention into thecells. In one embodiment of the present invention, the peptide is partof a fusion protein which comprises, for example, the 80 N-terminalamino acids of the HLA-DR antigen-associated invariant chain (p33, inthe following “Ii”) as derived from the NCBI, GenBank Accession numberX00497. In other fusions, the peptides of the present invention can befused to an antibody as described herein, or a functional part thereof,in particular into a sequence of an antibody, so as to be specificallytargeted by said antibody, or, for example, to or into an antibody thatis specific for dendritic cells as described herein.

In addition, the peptide or variant may be modified further to improvestability and/or binding to MHC molecules in order to elicit a strongerimmune response. Methods for such an optimization of a peptide sequenceare well known in the art and include, for example, the introduction ofreverse peptide bonds or non-peptide bonds.

In a reverse peptide bond amino acid, residues are not joined by peptide(—CO—NN—) linkages but the peptide bond is reversed. Such retro-inversopeptidomimetics may be made using methods known in the art, for examplesuch as those described in Meziere et al (1997) (Meziere et al., 1997),incorporated herein by reference. This approach involves makingpseudopeptides containing changes involving the backbone, and not theorientation of side chains. Meziere et al. (Meziere et al., 1997) showthat for MHC binding and T helper cell responses, these pseudopeptidesare useful. Retro-inverse peptides, which contain NH—CO bonds instead ofCO—NH peptide bonds, are much more resistant to proteolysis.

A non-peptide bond is, for example, —CH₂—NH, —CH₂S—, —CH₂CH₂—, —CH═CH—,—COCH₂—, —CH(OH)CH₂—, and —CH₂SO—. U.S. Pat. No. 4,897,445 provides amethod for the solid phase synthesis of non-peptide bonds (—CH₂—NH) inpolypeptide chains which involves polypeptides synthesized by standardprocedures and the non-peptide bond synthesized by reacting an aminoaldehyde and an amino acid in the presence of NaCNBH₃.

Peptides comprising the sequences described above may be synthesizedwith additional chemical groups present at their amino and/or carboxytermini, to enhance the stability, bioavailability, and/or affinity ofthe peptides. For example, hydrophobic groups such as carbobenzoxyl,dansyl, or t-butyloxycarbonyl groups may be added to the peptides' aminotermini. Likewise, an acetyl group or a 9-fluorenylmethoxy-carbonylgroup may be placed at the peptides' amino termini. Additionally, thehydrophobic group, t-butyloxycarbonyl, or an amido group may be added tothe peptides' carboxy termini.

Further, the peptides of the invention may be synthesized to alter theirsteric configuration. For example, the D-isomer of one or more of theamino acid residues of the peptide may be used, rather than the usualL-isomer. Still further, at least one of the amino acid residues of thepeptides of the invention may be substituted by one of the well-knownnon-naturally occurring amino acid residues. Alterations such as thesemay serve to increase the stability, bioavailability and/or bindingaction of the peptides of the invention.

Similarly, a peptide or variant of the invention may be modifiedchemically by reacting specific amino acids either before or aftersynthesis of the peptide. Examples for such modifications are well knownin the art and are summarized e.g. in R. Lundblad, Chemical Reagents forProtein Modification, 3rd ed. CRC Press, 2004(Lundblad, 2004), which isincorporated herein by reference. Chemical modification of amino acidsincludes but is not limited to, modification by acylation, amidination,pyridoxylation of lysine, reductive alkylation, trinitrobenzylation ofamino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS), amidemodification of carboxyl groups and sulphydryl modification by performicacid oxidation of cysteine to cysteic acid, formation of mercurialderivatives, formation of mixed disulphides with other thiol compounds,reaction with maleimide, carboxymethylation with iodoacetic acid oriodoacetamide and carbamoylation with cyanate at alkaline pH, althoughwithout limitation thereto. In this regard, the skilled person isreferred to Chapter 15 of Current Protocols In Protein Science, Eds.Coligan et al. (John Wiley and Sons NY 1995-2000) (Coligan et al., 1995)for more extensive methodology relating to chemical modification ofproteins.

Briefly, modification of e.g. arginyl residues in proteins is oftenbased on the reaction of vicinal dicarbonyl compounds such asphenylglyoxal, 2,3-butanedione, and 1,2-cyclohexanedione to form anadduct. Another example is the reaction of methylglyoxal with arginineresidues. Cysteine can be modified without concomitant modification ofother nucleophilic sites such as lysine and histidine. As a result, alarge number of reagents are available for the modification of cysteine.The websites of companies such as Sigma-Aldrich(http://www.sigma-aldrich.com) provide information on specific reagents.

Selective reduction of disulfide bonds in proteins is also common.Disulfide bonds can be formed and oxidized during the heat treatment ofbiopharmaceuticals. Woodward's Reagent K may be used to modify specificglutamic acid residues. N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimidecan be used to form intra-molecular crosslinks between a lysine residueand a glutamic acid residue. For example, diethylpyrocarbonate is areagent for the modification of histidyl residues in proteins. Histidinecan also be modified using 4-hydroxy-2-nonenal. The reaction of lysineresidues and other α-amino groups is, for example, useful in binding ofpeptides to surfaces or the cross-linking of proteins/peptides. Lysineis the site of attachment of poly(ethylene)glycol and the major site ofmodification in the glycosylation of proteins. Methionine residues inproteins can be modified with e.g. iodoacetamide, bromoethylamine, andchloramine T.

Tetranitromethane and N-acetylimidazole can be used for the modificationof tyrosyl residues. Cross-linking via the formation of dityrosine canbe accomplished with hydrogen peroxide/copper ions.

Recent studies on the modification of tryptophan have usedN-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide or3-bromo-3-methyl-2-(2-nitrophenylmercapto)-3H-indole (BPNS-skatole).

Successful modification of therapeutic proteins and peptides with PEG isoften associated with an extension of circulatory half-life whilecross-linking of proteins with glutaraldehyde, polyethylene glycoldiacrylate and formaldehyde is used for the preparation of hydrogels.Chemical modification of allergens for immunotherapy is often achievedby carbamylation with potassium cyanate.

A peptide or variant, wherein the peptide is modified or includesnon-peptide bonds is a preferred embodiment of the invention.

Another embodiment of the present invention relates to a non-naturallyoccurring peptide wherein said peptide consists or consists essentiallyof an amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 398and has been synthetically produced (e.g. synthesized) as apharmaceutically acceptable salt. Methods to synthetically producepeptides are well known in the art. The salts of the peptides accordingto the present invention differ substantially from the peptides in theirstate(s) in vivo, as the peptides as generated in vivo are no salts. Thenon-natural salt form of the peptide mediates the solubility of thepeptide, in particular in the context of pharmaceutical compositionscomprising the peptides, e.g. the peptide vaccines as disclosed herein.A sufficient and at least substantial solubility of the peptide(s) isrequired in order to efficiently provide the peptides to the subject tobe treated. Preferably, the salts are pharmaceutically acceptable saltsof the peptides. These salts according to the invention include alkalineand earth alkaline salts such as salts of the Hofmeister seriescomprising as anions PO₄ ³⁻, SO₄ ²⁻, CH₃COO⁻, Cl⁻, Br⁻, NO₃ ⁻, ClO₄ ⁻,I⁻, SCN⁻ and as cations NH₄ ⁺, Rb⁺, K⁺, Na⁺, Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺,Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts are selected from (NH₄)₃PO₄,(NH₄)₂HPO₄, (NH₄)H₂PO₄, (NH₄)₂SO₄, NH₄CH₃COO, NH₄Cl, NH₄Br, NH₄NO₃,NH₄ClO₄, NH₄₁, NH₄SCN, Rb₃PO₄, Rb₂HPO₄, RbH₂PO₄, Rb₂SO₄, Rb₄CH₃COO,Rb₄Cl, Rb₄Br, Rb₄NO₃, Rb₄ClO₄, Rb₄₁, Rb₄SCN, K₃PO₄, K₂HPO₄, KH₂PO₄,K₂SO₄, KCH₃COO, KCl, KBr, KNO₃, KClO₄, KI, KSCN, Na₃PO₄, Na₂HPO₄,NaH₂PO₄, Na₂SO₄, NaCH₃COO, NaCl, NaBr, NaNO₃, NaClO₄, NaI, NaSCN, ZnCl₂Cs₃PO₄, Cs₂HPO₄, CsH₂PO₄, Cs₂SO₄, CsCH₃COO, CsCl, CsBr, CsNO₃, CSClO₄,CsI, CsSCN, Li₃PO₄, Li₂HPO₄, LiH₂PO₄, Li₂SO₄, LiCH₃COO, LiCl, LiBr,LiNO₃, LiClO₄, LiI, LiSCN, Cu₂SO₄, Mg₃(PO₄)₂, Mg₂HPO₄, Mg(H₂PO₄)₂,Mg₂SO₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(NO₃)₂, Mg(ClO₄)₂, MgI₂, Mg(SCN)₂,MnC₂, Ca₃(PO₄), Ca₂HPO₄, Ca(H₂PO₄)₂, CaSO₄, Ca(CH₃COO)₂, CaCl₂), CaBr₂,Ca(N₀₃)₂, Ca(ClO₄)₂, CaI₂, Ca(SCN)₂, Ba₃(PO₄)₂, Ba₂HPO₄, Ba(H₂PO₄)₂,BaSO₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(N₀₃)₂, Ba(ClO₄)₂, Ba₂, andBa(SCN)₂. Particularly preferred are NH acetate, MgCl₂, KH₂PO₄, Na₂SO₄,KCl, NaCl, and CaCl₂, such as, for example, the chloride or acetate(trifluoroacetate) salts.

Generally, peptides and variants (at least those containing peptidelinkages between amino acid residues) may be synthesized by theFmoc-polyamide mode of solid-phase peptide synthesis as disclosed byLukas et al. (Lukas et al., 1981), and by references as cited therein.Temporary N-amino group protection is afforded by the9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of thishighly base-labile protecting group is done using 20% piperidine inN,N-dimethylformamide. Side-chain functionalities may be protected astheir butyl ethers (in the case of serine threonine and tyrosine), butylesters (in the case of glutamic acid and aspartic acid),butyloxycarbonyl derivative (in the case of lysine and histidine),trityl derivative (in the case of cysteine) and4-methoxy-2,3,6-trimethylbenzenesulphonyl derivative (in the case ofarginine). Where glutamine or asparagine are C-terminal residues, use ismade of the 4,4′-dimethoxybenzhydryl group for protection of the sidechain amido functionalities. The solid-phase support is based on apolydimethyl-acrylamide polymer constituted from the three monomersdimethylacrylamide (backbone-monomer), bisacryloylethylene diamine(cross linker) and acryloylsarcosine methyl ester (functionalizingagent). The peptide-to-resin cleavable linked agent used is theacid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All aminoacid derivatives are added as their preformed symmetrical anhydridederivatives with the exception of asparagine and glutamine, which areadded using a reversed N, N-dicyclohexyl-carbodiimide/1hydroxybenzotriazole mediated coupling procedure. All coupling anddeprotection reactions are monitored using ninhydrin, trinitrobenzenesulphonic acid or isotin test procedures. Upon completion of synthesis,peptides are cleaved from the resin support with concomitant removal ofside-chain protecting groups by treatment with 95% trifluoroacetic acidcontaining a 50% scavenger mix. Scavengers commonly used includeethanedithiol, phenol, anisole and water, the exact choice depending onthe constituent amino acids of the peptide being synthesized. Also acombination of solid phase and solution phase methodologies for thesynthesis of peptides is possible (see, for example, (Bruckdorfer etal., 2004), and the references as cited therein).

Trifluoroacetic acid is removed by evaporation in vacuo, with subsequenttrituration with diethyl ether affording the crude peptide. Anyscavengers present are removed by a simple extraction procedure which onlyophilization of the aqueous phase affords the crude peptide free ofscavengers. Reagents for peptide synthesis are generally available frome.g. Calbiochem-Novabiochem (Nottingham, UK).

Purification may be performed by anyone, or a combination of, techniquessuch as re-crystallization, size exclusion chromatography, ion-exchangechromatography, hydrophobic interaction chromatography and (usually)reverse-phase high performance liquid chromatography using e.g.acetonitrile/water gradient separation.

Analysis of peptides may be carried out using thin layer chromatography,electrophoresis, in particular capillary electrophoresis, solid phaseextraction (CSPE), reverse-phase high performance liquid chromatography,amino-acid analysis after acid hydrolysis and by fast atom bombardment(FAB) mass spectrometric analysis, as well as MALDI and ESI-Q-TOF massspectrometric analysis.

In order to select over-presented peptides, a presentation profile iscalculated showing the median sample presentation as well as replicatevariation. The profile juxtaposes samples of the tumor entity ofinterest to a baseline of normal tissue samples. Each of these profilescan then be consolidated into an over-presentation score by calculatingthe p-value of a Linear Mixed-Effects Model (Pinheiro et al., 2015)adjusting for multiple testing by False Discovery Rate (Benjamini andHochberg, 1995) (cf. Example 1, FIGS. 1A-1N).

For the identification and relative quantitation of HLA ligands by massspectrometry, HLA molecules from shock-frozen tissue samples werepurified and HLA-associated peptides were isolated. The isolatedpeptides were separated and sequences were identified by onlinenano-electrospray-ionization (nanoESI) liquid chromatography-massspectrometry (LC-MS) experiments. The resulting peptide sequences wereverified by comparison of the fragmentation pattern of naturaltumor-associated peptides (TUMAPs) recorded from acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer samples (N=155samples) with the fragmentation patterns of corresponding syntheticreference peptides of identical sequences. Since the peptides weredirectly identified as ligands of HLA molecules of primary tumors, theseresults provide direct evidence for the natural processing andpresentation of the identified peptides on primary cancer tissueobtained from 155 acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer patients.

The discovery pipeline XPRESIDENT® v2.1 (see, for example, US2013-0096016, which is hereby incorporated by reference in its entirety)allows the identification and selection of relevant over-presentedpeptide vaccine candidates based on direct relative quantitation ofHLA-restricted peptide levels on cancer tissues in comparison to severaldifferent non-cancerous tissues and organs. This was achieved by thedevelopment of label-free differential quantitation using the acquiredLC-MS data processed by a proprietary data analysis pipeline, combiningalgorithms for sequence identification, spectral clustering, ioncounting, retention time alignment, charge state deconvolution andnormalization.

Presentation levels including error estimates for each peptide andsample were established. Peptides exclusively presented on tumor tissueand peptides over-presented in tumor versus non-cancerous tissues andorgans have been identified.

HLA-peptide complexes from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer tissue samples were purified andHLA-associated peptides were isolated and analyzed by LC-MS (see example1). All TUMAPs contained in the present application were identified withthis approach on acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer samples confirming their presentation onacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

TUMAPs identified on multiple acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer and normal tissues were quantified usingion-counting of label-free LC-MS data. The method assumes that LC-MSsignal areas of a peptide correlate with its abundance in the sample.All quantitative signals of a peptide in various LC-MS experiments werenormalized based on central tendency, averaged per sample and mergedinto a bar plot, called presentation profile. The presentation profileconsolidates different analysis methods like protein database search,spectral clustering, charge state deconvolution (decharging) andretention time alignment and normalization.

Besides over-presentation of the peptide, mRNA expression of theunderlying gene was tested. mRNA data were obtained via RNASeq analysesof normal tissues and cancer tissues (cf. Example 2, FIGS. 2A-2P). Anadditional source of normal tissue data was a database of publiclyavailable RNA expression data from around 3000 normal tissue samples(Lonsdale, 2013). Peptides which are derived from proteins whose codingmRNA is highly expressed in cancer tissue, but very low or absent invital normal tissues, were preferably included in the present invention.

The present invention provides peptides that are useful in treatingcancers/tumors, preferably acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer that over- or exclusively present thepeptides of the invention. These peptides were shown by massspectrometry to be naturally presented by HLA molecules on primary humanacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancersamples.

Many of the source gene/proteins (also designated “full-length proteins”or “underlying proteins”) from which the peptides are derived were shownto be highly over-expressed in cancer compared with normaltissues—“normal tissues” in relation to this invention shall mean eitherhealthy blood cells, blood vessels, brain, heart, liver, lung, bileduct, bladder, bone marrow, esophagus, large intestine, kidney,peripheral nerve, pancreas, skin, spinal cord, spleen, stomach, thyroid,trachea cells or other normal tissue cells, demonstrating a high degreeof tumor association of the source genes (see Example 2). Moreover, thepeptides themselves are strongly over-presented on tumor tissue—“tumortissue” in relation to this invention shall mean a sample from a patientsuffering from acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lungcancer (including non-small cell lung cancer adenocarcinoma, squamouscell non-small cell lung cancer, and small cell lung cancer), ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, urinary bladder carcinoma, uterine and endometrialcancer, but not on normal tissues (see Example 1).

HLA-bound peptides can be recognized by the immune system, specificallyT lymphocytes. T cells can destroy the cells presenting the recognizedHLA/peptide complex, e.g. acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer cells presenting the derived peptides.

The peptides of the present invention have been shown to be capable ofstimulating T cell responses and/or are over-presented and thus can beused for the production of antibodies and/or TCRs, such as soluble TCRs,according to the present invention (see Example 3, Example 4).Furthermore, the peptides when complexed with the respective MHC can beused for the production of antibodies and/or TCRs, in particular sTCRs,according to the present invention, as well. Respective methods are wellknown to the person of skill and can be found in the respectiveliterature as well (see also below). Thus, the peptides of the presentinvention are useful for generating an immune response in a patient bywhich tumor cells can be destroyed. An immune response in a patient canbe induced by direct administration of the described peptides orsuitable precursor substances (e.g. elongated peptides, proteins, ornucleic acids encoding these peptides) to the patient, ideally incombination with an agent enhancing the immunogenicity (i.e. anadjuvant). The immune response originating from such a therapeuticvaccination can be expected to be highly specific against tumor cellsbecause the target peptides of the present invention are not presentedon normal tissues in comparable copy numbers, preventing the risk ofundesired autoimmune reactions against normal cells in the patient.

The present description further relates to T-cell receptors (TCRs)comprising an alpha chain and a beta chain (“alpha/beta TCRs”). Alsoprovided are peptides according to the invention capable of binding toTCRs and antibodies when presented by an MHC molecule.

The present description also relates to fragments of the TCRs accordingto the invention that are capable of binding to a peptide antigenaccording to the present invention when presented by an HLA molecule.The term particularly relates to soluble TCR fragments, for example TCRsmissing the transmembrane parts and/or constant regions, single chainTCRs, and fusions thereof to, for example, with Ig.

The present description also relates to nucleic acids, vectors and hostcells for expressing TCRs and peptides of the present description; andmethods of using the same.

The term “T-cell receptor” (abbreviated TCR) refers to a heterodimericmolecule comprising an alpha polypeptide chain (alpha chain) and a betapolypeptide chain (beta chain), wherein the heterodimeric receptor iscapable of binding to a peptide antigen presented by an HLA molecule.The term also includes so-called gamma/delta TCRs.

In one embodiment the description provides a method of producing a TCRas described herein, the method comprising culturing a host cell capableof expressing the TCR under conditions suitable to promote expression ofthe TCR.

The description in another aspect relates to methods according to thedescription, wherein the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellor artificial antigen-presenting cell by contacting a sufficient amountof the antigen with an antigen-presenting cell or the antigen is loadedonto class I or II MHC tetramers by tetramerizing the antigen/class I orII MHC complex monomers.

The alpha and beta chains of alpha/beta TCR's, and the gamma and deltachains of gamma/delta TCRs, are generally regarded as each having two“domains”, namely variable and constant domains. The variable domainconsists of a concatenation of variable region (V) and joining region(J). The variable domain may also include a leader region (L). Beta anddelta chains may also include a diversity region (D). The alpha and betaconstant domains may also include C-terminal transmembrane (TM) domainsthat anchor the alpha and beta chains to the cell membrane.

With respect to gamma/delta TCRs, the term “TCR gamma variable domain”as used herein refers to the concatenation of the TCR gamma V (TRGV)region without leader region (L), and the TCR gamma J (TRGJ) region, andthe term TCR gamma constant domain refers to the extracellular TRGCregion, or to a C-terminal truncated TRGC sequence. Likewise, the term“TCR delta variable domain” refers to the concatenation of the TCR deltaV (TRDV) region without leader region (L) and the TCR delta D/J(TRDD/TRDJ) region, and the term “TCR delta constant domain” refers tothe extracellular TRDC region, or to a C-terminal truncated TRDCsequence.

TCRs of the present description preferably bind to a peptide-HLAmolecule complex with a binding affinity (KD) of about 100 μM or less,about 50 μM or less, about 25 μM or less, or about 10 μM or less. Morepreferred are high affinity TCRs having binding affinities of about 1 μMor less, about 100 nM or less, about 50 nM or less, about 25 nM or less.Non-limiting examples of preferred binding affinity ranges for TCRs ofthe present invention include about 1 nM to about 10 nM; about 10 nM toabout 20 nM; about 20 nM to about 30 nM; about 30 nM to about 40 nM;about 40 nM to about 50 nM; about 50 nM to about 60 nM; about 60 nM toabout 70 nM; about 70 nM to about 80 nM; about 80 nM to about 90 nM; andabout 90 nM to about 100 nM.

As used herein in connect with TCRs of the present description,“specific binding” and grammatical variants thereof are used to mean aTCR having a binding affinity (KD) for a peptide-HLA molecule complex of100 μM or less.

Alpha/beta heterodimeric TCRs of the present description may have anintroduced disulfide bond between their constant domains. Preferred TCRsof this type include those which have a TRAC constant domain sequenceand a TRBC1 or TRBC2 constant domain sequence except that Thr 48 of TRACand Ser 57 of TRBC1 or TRBC2 are replaced by cysteine residues, the saidcysteines forming a disulfide bond between the TRAC constant domainsequence and the TRBC1 or TRBC2 constant domain sequence of the TCR.

With or without the introduced inter-chain bond mentioned above,alpha/beta hetero-dimeric TCRs of the present description may have aTRAC constant domain sequence and a TRBC1 or TRBC2 constant domainsequence, and the TRAC constant domain sequence and the TRBC1 or TRBC2constant domain sequence of the TCR may be linked by the nativedisulfide bond between Cys4 of exon 2 of TRAC and Cys2 of exon 2 ofTRBC1 or TRBC2.

TCRs of the present description may comprise a detectable label selectedfrom the group consisting of a radionuclide, a fluorophore and biotin.TCRs of the present description may be conjugated to a therapeuticallyactive agent, such as a radionuclide, a chemotherapeutic agent, or atoxin.

In an embodiment, a TCR of the present description having at least onemutation in the alpha chain and/or having at least one mutation in thebeta chain has modified glycosylation compared to the unmutated TCR.

In an embodiment, a TCR comprising at least one mutation in the TCRalpha chain and/or TCR beta chain has a binding affinity for, and/or abinding half-life for, a peptide-HLA molecule complex, which is at leastdouble that of a TCR comprising the unmutated TCR alpha chain and/orunmutated TCR beta chain. Affinity-enhancement of tumor-specific TCRs,and its exploitation, relies on the existence of a window for optimalTCR affinities. The existence of such a window is based on observationsthat TCRs specific for HLA-A1-restricted pathogens have KD values thatare generally about 10-fold lower when compared to TCRs specific forHLA-A1-restricted tumor-associated self-antigens (this holds true forother alleles as well). It is now known, although tumor antigens havethe potential to be immunogenic, because tumors arise from theindividual's own cells only mutated proteins or proteins with alteredtranslational processing will be seen as foreign by the immune system.Antigens that are upregulated or overexpressed (so called self-antigens)will not necessarily induce a functional immune response against thetumor: T-cells expressing TCRs that are highly reactive to theseantigens will have been negatively selected within the thymus in aprocess known as central tolerance, meaning that only T-cells withlow-affinity TCRs for self-antigens remain. Therefore, affinity of TCRsor variants of the present description to peptides can be enhanced bymethods well known in the art.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising incubating PBMCs from HLA-A*01-negative healthy donors withA*01/peptide monomers, incubating the PBMCs with tetramer-phycoerythrin(PE) and isolating the high avidity T-cells by fluorescence activatedcell sorting (FACS)-Calibur analysis.

The present description further relates to a method of identifying andisolating a TCR according to the present description, said methodcomprising obtaining a transgenic mouse with the entire human TCRαβ geneloci (1.1 and 0.7 Mb), whose T-cells express a diverse human TCRrepertoire that compensates for mouse TCR deficiency, immunizing themouse with a peptide, incubating PBMCs obtained from the transgenic micewith tetramer-phycoerythrin (PE), and isolating the high avidity T-cellsby fluorescence activated cell sorting (FACS)-Calibur analysis.

In one aspect, to obtain T-cells expressing TCRs of the presentdescription, nucleic acids encoding TCR-alpha and/or TCR-beta chains ofthe present description are cloned into expression vectors, such asgamma retrovirus or lentivirus. The recombinant viruses are generatedand then tested for functionality, such as antigen specificity andfunctional avidity. An aliquot of the final product is then used totransduce the target T-cell population (generally purified from patientPBMCs), which is expanded before infusion into the patient.

In another aspect, to obtain T-cells expressing TCRs of the presentdescription, TCR RNAs are synthesized by techniques known in the art,e.g., in vitro transcription systems. The in vitro-synthesized TCR RNAsare then introduced into primary CD8+ T-cells obtained from healthydonors by electroporation to re-express tumor specific TCR-alpha and/orTCR-beta chains.

To increase the expression, nucleic acids encoding TCRs of the presentdescription may be operably linked to strong promoters, such asretroviral long terminal repeats (LTRs), cytomegalovirus (CMV), murinestem cell virus (MSCV) U3, phosphoglycerate kinase (PGK), β-actin,ubiquitin, and a simian virus 40 (SV40)/CD43 composite promoter,elongation factor (EF)-1a and the spleen focus-forming virus (SFFV)promoter. In a preferred embodiment, the promoter is heterologous to thenucleic acid being expressed.

In addition to strong promoters, TCR expression cassettes of the presentdescription may contain additional elements that can enhance transgeneexpression, including a central polypurine tract (cPPT), which promotesthe nuclear translocation of lentiviral constructs (Follenzi et al.,2000), and the woodchuck hepatitis virus posttranscriptional regulatoryelement (wPRE), which increases the level of transgene expression byincreasing RNA stability (Zufferey et al., 1999).

The alpha and beta chains of a TCR of the present invention may beencoded by nucleic acids located in separate vectors or may be encodedby polynucleotides located in the same vector.

Achieving high-level TCR surface expression requires that both theTCR-alpha and TCR-beta chains of the introduced TCR be transcribed athigh levels. To do so, the TCR-alpha and TCR-beta chains of the presentdescription may be cloned into bi-cistronic constructs in a singlevector, which has been shown to be capable of over-coming this obstacle.The use of a viral intraribosomal entry site (IRES) between theTCR-alpha and TCR-beta chains results in the coordinated expression ofboth chains, because the TCR-alpha and TCR-beta chains are generatedfrom a single transcript that is broken into two proteins duringtranslation, ensuring that an equal molar ratio of TCR-alpha andTCR-beta chains are produced (Schmitt et al., 2009).

Nucleic acids encoding TCRs of the present description may be codonoptimized to increase expression from a host cell. Redundancy in thegenetic code allows some amino acids to be encoded by more than onecodon, but certain codons are less “optimal” than others because of therelative availability of matching tRNAs as well as other factors(Gustafsson et al., 2004). Modifying the TCR-alpha and TCR-beta genesequences such that each amino acid is encoded by the optimal codon formammalian gene expression, as well as eliminating mRNA instabilitymotifs or cryptic splice sites, has been shown to significantly enhanceTCR-alpha and TCR-beta gene expression (Scholten et al., 2006).

Furthermore, mispairing between the introduced and endogenous TCR chainsmay result in the acquisition of specificities that pose a significantrisk for autoimmunity. For example, the formation of mixed TCR dimersmay reduce the number of CD3 molecules available to form properly pairedTCR complexes, and therefore can significantly decrease the functionalavidity of the cells expressing the introduced TCR (Kuball et al.,2007).

To reduce mispairing, the C-terminus domain of the introduced TCR chainsof the present description may be modified in order to promoteinterchain affinity, while de-creasing the ability of the introducedchains to pair with the endogenous TCR. These strategies may includereplacing the human TCR-alpha and TCR-beta C-terminus domains with theirmurine counterparts (murinized C-terminus domain); generating a secondinterchain disulfide bond in the C-terminus domain by introducing asecond cysteine residue into both the TCR-alpha and TCR-beta chains ofthe introduced TCR (cysteine modification); swapping interactingresidues in the TCR-alpha and TCR-beta chain C-terminus domains(“knob-in-hole”); and fusing the variable domains of the TCR-alpha andTCR-beta chains directly to CD3ζ (CD3ζ fusion) (Schmitt et al., 2009).

In an embodiment, a host cell is engineered to express a TCR of thepresent description. In preferred embodiments, the host cell is a humanT-cell or T-cell progenitor. In some embodiments the T-cell or T-cellprogenitor is obtained from a cancer patient. In other embodiments theT-cell or T-cell progenitor is obtained from a healthy donor. Host cellsof the present description can be allogeneic or autologous with respectto a patient to be treated. In one embodiment, the host is a gamma/deltaT-cell transformed to express an alpha/beta TCR.

A “pharmaceutical composition” is a composition suitable foradministration to a human being in a medical setting. Preferably, apharmaceutical composition is sterile and produced according to GMPguidelines.

The pharmaceutical compositions comprise the peptides either in the freeform or in the form of a pharmaceutically acceptable salt (see alsoabove). As used herein, “a pharmaceutically acceptable salt” refers to aderivative of the disclosed peptides wherein the peptide is modified bymaking acid or base salts of the agent. For example, acid salts areprepared from the free base (typically wherein the neutral form of thedrug has a neutral —NH2 group) involving reaction with a suitable acid.Suitable acids for preparing acid salts include both organic acids,e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, malic acid, malonic acid, succinic acid, maleic acid, fumaricacid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelicacid, methane sulfonic acid, ethane sulfonic acid, p-toluenesulfonicacid, salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acidphosphoric acid and the like. Conversely, preparation of basic salts ofacid moieties which may be present on a peptide are prepared using apharmaceutically acceptable base such as sodium hydroxide, potassiumhydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or thelike.

In an especially preferred embodiment, the pharmaceutical compositionscomprise the peptides as salts of acetic acid (acetates), trifluoroacetates or hydrochloric acid (chlorides).

Preferably, the medicament of the present invention is animmunotherapeutic such as a vaccine. It may be administered directlyinto the patient, into the affected organ or systemically i.d., i.m.,s.c., i.p. and i.v., or applied ex vivo to cells derived from thepatient or a human cell line which are subsequently administered to thepatient or used in vitro to select a subpopulation of immune cellsderived from the patient, which are then re-administered to the patient.If the nucleic acid is administered to cells in vitro, it may be usefulfor the cells to be transfected so as to co-express immune-stimulatingcytokines, such as interleukin-2. The peptide may be substantially pureor combined with an immune-stimulating adjuvant (see below) or used incombination with immune-stimulatory cytokines, or be administered with asuitable delivery system, for example liposomes. The peptide may also beconjugated to a suitable carrier such as keyhole limpet haemocyanin(KLH) or mannan (see WO 95/18145 and (Longenecker et al., 1993)). Thepeptide may also be tagged, may be a fusion protein, or may be a hybridmolecule. The peptides whose sequence is given in the present inventionare expected to stimulate CD4 or CD8 T cells. However, stimulation ofCD8 T cells is more efficient in the presence of help provided by

CD4 T-helper cells. Thus, for MHC Class I epitopes that stimulate CD8 Tcells the fusion partner or sections of a hybrid molecule suitablyprovide epitopes which stimulate CD4-positive T cells. CD4- andCD8-stimulating epitopes are well known in the art and include thoseidentified in the present invention.

In one aspect, the vaccine comprises at least one peptide having theamino acid sequence set forth SEQ ID No. 1 to SEQ ID No. 398, and atleast one additional peptide, preferably two to 50, more preferably twoto 25, even more preferably two to 20 and most preferably two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, fifteen, sixteen, seventeen or eighteen peptides. Thepeptide(s) may be derived from one or more specific TAAs and may bind toMHC class I molecules.

A further aspect of the invention provides a nucleic acid (for example apolynucleotide) encoding a peptide or peptide variant of the invention.The polynucleotide may be, for example, DNA, cDNA, PNA, RNA orcombinations thereof, either single- and/or double-stranded, or nativeor stabilized forms of polynucleotides, such as, for example,polynucleotides with a phosphorothioate backbone and it may or may notcontain introns so long as it codes for the peptide. Of course, onlypeptides that contain naturally occurring amino acid residues joined bynaturally occurring peptide bonds are encodable by a polynucleotide. Astill further aspect of the invention provides an expression vectorcapable of expressing a polypeptide according to the invention.

A variety of methods have been developed to link polynucleotides,especially DNA, to vectors for example via complementary cohesivetermini. For instance, complementary homopolymer tracts can be added tothe DNA segment to be inserted to the vector DNA. The vector and DNAsegment are then joined by hydrogen bonding between the complementaryhomopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide analternative method of joining the DNA segment to vectors. Syntheticlinkers containing a variety of restriction endonuclease sites arecommercially available from a number of sources including InternationalBiotechnologies Inc. New Haven, Conn., USA.

A desirable method of modifying the DNA encoding the polypeptide of theinvention employs the polymerase chain reaction as disclosed by Saiki RK, et al. (Saiki et al., 1988). This method may be used for introducingthe DNA into a suitable vector, for example by engineering in suitablerestriction sites, or it may be used to modify the DNA in other usefulways as is known in the art. If viral vectors are used, pox- oradenovirus vectors are preferred.

The DNA (or in the case of retroviral vectors, RNA) may then beexpressed in a suitable host to produce a polypeptide comprising thepeptide or variant of the invention. Thus, the DNA encoding the peptideor variant of the invention may be used in accordance with knowntechniques, appropriately modified in view of the teachings containedherein, to construct an expression vector, which is then used totransform an appropriate host cell for the expression and production ofthe polypeptide of the invention. Such techniques include thosedisclosed, for example, in U.S. Pat. Nos. 4,440,859, 4,530,901,4,582,800, 4,677,063, 4,678,751, 4,704,362, 4,710,463, 4,757,006,4,766,075, and 4,810,648.

The DNA (or in the case of retroviral vectors, RNA) encoding thepolypeptide constituting the compound of the invention may be joined toa wide variety of other DNA sequences for introduction into anappropriate host. The companion DNA will depend upon the nature of thehost, the manner of the introduction of the DNA into the host, andwhether episomal maintenance or integration is desired.

Generally, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognized bythe desired host, although such controls are generally available in theexpression vector. The vector is then introduced into the host throughstandard techniques. Generally, not all of the hosts will be transformedby the vector. Therefore, it will be necessary to select for transformedhost cells. One selection technique involves incorporating into theexpression vector a DNA sequence, with any necessary control elements,that codes for a selectable trait in the transformed cell, such asantibiotic resistance.

Alternatively, the gene for such selectable trait can be on anothervector, which is used to co-transform the desired host cell.

Host cells that have been transformed by the recombinant DNA of theinvention are then cultured for a sufficient time and under appropriateconditions known to those skilled in the art in view of the teachingsdisclosed herein to permit the expression of the polypeptide, which canthen be recovered.

Many expression systems are known, including bacteria (for example E.coli and Bacillus subtilis), yeasts (for example Saccharomycescerevisiae), filamentous fungi (for example Aspergillus spec.), plantcells, animal cells and insect cells. Preferably, the system can bemammalian cells such as CHO cells available from the ATCC Cell BiologyCollection.

A typical mammalian cell vector plasmid for constitutive expressioncomprises the CMV or SV40 promoter with a suitable poly A tail and aresistance marker, such as neomycin. One example is pSVL available fromPharmacia, Piscataway, N.J., USA. An example of an inducible mammalianexpression vector is pMSG, also available from Pharmacia. Useful yeastplasmid vectors are pRS403-406 and pRS413-416 and are generallyavailable from Stratagene Cloning Systems, La Jolla, Calif. 92037, USA.Plasmids pRS403, pRS404, pRS405 and pRS406 are Yeast Integratingplasmids (Ylps) and incorporate the yeast selectable markers HIS3, TRP1,LEU2 and URA3. Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).CMV promoter-based vectors (for example from Sigma-Aldrich) providetransient or stable expression, cytoplasmic expression or secretion, andN-terminal or C-terminal tagging in various combinations of FLAG,3×FLAG, c-myc or MAT. These fusion proteins allow for detection,purification and analysis of recombinant protein. Dual-tagged fusionsprovide flexibility in detection.

The strong human cytomegalovirus (CMV) promoter regulatory region drivesconstitutive protein expression levels as high as 1 mg/L in COS cells.For less potent cell lines, protein levels are typically ˜0.1 mg/L. Thepresence of the SV40 replication origin will result in high levels ofDNA replication in SV40 replication permissive COS cells. CMV vectors,for example, can contain the pMB1 (derivative of pBR322) origin forreplication in bacterial cells, the b-lactamase gene for ampicillinresistance selection in bacteria, hGH polyA, and the f1 origin. Vectorscontaining the pre-pro-trypsin leader (PPT) sequence can direct thesecretion of FLAG fusion proteins into the culture medium forpurification using ANTI-FLAG antibodies, resins, and plates. Othervectors and expression systems are well known in the art for use with avariety of host cells.

In another embodiment two or more peptides or peptide variants of theinvention are encoded and thus expressed in a successive order (similarto “beads on a string” constructs). In doing so, the peptides or peptidevariants may be linked or fused together by stretches of linker aminoacids, such as for example LLLLLL, or may be linked without anyadditional peptide(s) between them. These constructs can also be usedfor cancer therapy and may induce immune responses both involving MHC Iand MHC II.

The present invention also relates to a host cell transformed with apolynucleotide vector construct of the present invention. The host cellcan be either prokaryotic or eukaryotic. Bacterial cells may bepreferred prokaryotic host cells in some circumstances and typically area strain of E. coli such as, for example, the E. coli strains DH5available from Bethesda Research Laboratories Inc., Bethesda, Md., USA,and RR1 available from the American Type Culture Collection (ATCC) ofRockville, Md., USA (No ATCC 31343). Preferred eukaryotic host cellsinclude yeast, insect and mammalian cells, preferably vertebrate cellssuch as those from a mouse, rat, monkey or human fibroblastic and coloncell lines. Yeast host cells include YPH499, YPH500 and YPH501, whichare generally available from Stratagene Cloning Systems, La Jolla,Calif. 92037, USA. Preferred mammalian host cells include Chinesehamster ovary (CHO) cells available from the ATCC as CCL61, NIH Swissmouse embryo cells NIH/3T3 available from the ATCC as CRL 1658, monkeykidney-derived COS-1 cells available from the ATCC as CRL 1650 and 293cells which are human embryonic kidney cells. Preferred insect cells areSf9 cells which can be transfected with baculovirus expression vectors.An overview regarding the choice of suitable host cells for expressioncan be found in, for example, the textbook of Paulina Balbas and ArgeliaLorence “Methods in Molecular Biology Recombinant Gene Expression,Reviews and Protocols,” Part One, Second Edition, ISBN978-1-58829-262-9, and other literature known to the person of skill.

Transformation of appropriate cell hosts with a DNA construct of thepresent invention is accomplished by well-known methods that typicallydepend on the type of vector used. With regard to transformation ofprokaryotic host cells, see, for example, Cohen et al. (Cohen et al.,1972) and (Green and Sambrook, 2012). Transformation of yeast cells isdescribed in Sherman et al. (Sherman et al., 1986). The method of Beggs(Beggs, 1978) is also useful. With regard to vertebrate cells, reagentsuseful in transfecting such cells, for example calcium phosphate andDEAE-dextran or liposome formulations, are available from StratageneCloning Systems, or Life Technologies Inc., Gaithersburg, Md. 20877,USA. Electroporation is also useful for transforming and/or transfectingcells and is well known in the art for transforming yeast cell,bacterial cells, insect cells and vertebrate cells.

Successfully transformed cells, i.e. cells that contain a DNA constructof the present invention, can be identified by well-known techniquessuch as PCR. Alternatively, the presence of the protein in thesupernatant can be detected using antibodies.

It will be appreciated that certain host cells of the invention areuseful in the preparation of the peptides of the invention, for examplebacterial, yeast and insect cells. However, other host cells may beuseful in certain therapeutic methods. For example, antigen-presentingcells, such as dendritic cells, may usefully be used to express thepeptides of the invention such that they may be loaded into appropriateMHC molecules. Thus, the current invention provides a host cellcomprising a nucleic acid or an expression vector according to theinvention.

In a preferred embodiment the host cell is an antigen presenting cell,in particular a dendritic cell or antigen presenting cell. APCs loadedwith a recombinant fusion protein containing prostatic acid phosphatase(PAP) were approved by the U.S. Food and Drug Administration (FDA) onApr. 29, 2010, to treat asymptomatic or minimally symptomatic metastaticHRPC (Sipuleucel-T) (Rini et al., 2006; Small et al., 2006).

A further aspect of the invention provides a method of producing apeptide or its variant, the method comprising culturing a host cell andisolating the peptide from the host cell or its culture medium.

In another embodiment, the peptide, the nucleic acid or the expressionvector of the invention are used in medicine. For example, the peptideor its variant may be prepared for intravenous (i.v.) injection,sub-cutaneous (s.c.) injection, intradermal (i.d.) injection,intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.Preferred methods of peptide injection include s.c., i.d., i.p., i.m.,and i.v. Preferred methods of DNA injection include i.d., i.m., s.c.,i.p. and i.v. Doses of e.g. between 50 μg and 1.5 mg, preferably 125 μgto 500 μg, of peptide or DNA may be given and will depend on therespective peptide or DNA. Dosages of this range were successfully usedin previous trials (Walter et al., 2012).

The polynucleotide used for active vaccination may be substantially pureor contained in a suitable vector or delivery system. The nucleic acidmay be DNA, cDNA, PNA, RNA or a combination thereof. Methods fordesigning and introducing such a nucleic acid are well known in the art.An overview is provided by e.g. Teufel et al. (Teufel et al., 2005).Polynucleotide vaccines are easy to prepare, but the mode of action ofthese vectors in inducing an immune response is not fully understood.Suitable vectors and delivery systems include viral DNA and/or RNA, suchas systems based on adenovirus, vaccinia virus, retroviruses, herpesvirus, adeno-associated virus or hybrids containing elements of morethan one virus. Non-viral delivery systems include cationic lipids andcationic polymers and are well known in the art of DNA delivery.Physical delivery, such as via a “gene-gun” may also be used. Thepeptide or peptides encoded by the nucleic acid may be a fusion protein,for example with an epitope that stimulates T cells for the respectiveopposite CDR as noted above.

The medicament of the invention may also include one or more adjuvants.Adjuvants are substances that non-specifically enhance or potentiate theimmune response (e.g., immune responses mediated by CD8-positive T cellsand helper-T (TH) cells to an antigen and would thus be considereduseful in the medicament of the present invention. Suitable adjuvantsinclude, but are not limited to, 1018 ISS, aluminum salts, AMPLIVAX®,AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, flagellin or TLR5 ligandsderived from flagellin, FLT3 ligand, GM-CSF, 1030, 1031, Imiquimod(ALDARA®), resiquimod, ImuFact IMP321, Interleukins as IL-2, IL-13,IL-21, Interferon-alpha or -beta, or pegylated derivatives thereof, ISPatch, ISS, ISCOMATRIX, ISCOMs, Juvlmmune®, LipoVac, MALP2, MF59,monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, MontanideISA 50V, Montanide ISA-51, water-in-oil and oil-in-water emulsions,OK-432, OM-174, OM-197-MP-EC, ONTAK, OspA, PepTel® vector system,poly(lactid co-glycolid) [PLG]-based and dextran microparticles,talactoferrin SRL172, Virosomes and other Virus-like particles, YF-17D,VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon, which isderived from saponin, mycobacterial extracts and synthetic bacterialcell wall mimics, and other proprietary adjuvants such as Ribi's Detox,Quil, or Superfos. Adjuvants such as Freund's or GM-CSF are preferred.Several immunological adjuvants (e.g., MF59) specific for dendriticcells and their preparation have been described previously (Allison andKrummel, 1995). Also, cytokines may be used. Several cytokines have beendirectly linked to influencing dendritic cell migration to lymphoidtissues (e.g., TNF-), accelerating the maturation of dendritic cellsinto efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF,IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporatedherein by reference in its entirety) and acting as immunoadjuvants(e.g., IL-12, IL-15, IL-23, IL-7, IFN-alpha. IFN-beta) (Gabrilovich etal., 1996).

CpG immunostimulatory oligonucleotides have also been reported toenhance the effects of adjuvants in a vaccine setting. Without beingbound by theory, CpG oligonucleotides act by activating the innate(non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.CpG triggered TLR9 activation enhances antigen-specific humoral andcellular responses to a wide variety of antigens, including peptide orprotein antigens, live or killed viruses, dendritic cell vaccines,autologous cellular vaccines and polysaccharide conjugates in bothprophylactic and therapeutic vaccines. More importantly it enhancesdendritic cell maturation and differentiation, resulting in enhancedactivation of TH1 cells and strong cytotoxic T-lymphocyte (CTL)generation, even in the absence of CD4 T cell help. The TH1 bias inducedby TLR9 stimulation is maintained even in the presence of vaccineadjuvants such as alum or incomplete Freund's adjuvant (IFA) thatnormally promote a TH2 bias. CpG oligonucleotides show even greateradjuvant activity when formulated or co-administered with otheradjuvants or in formulations such as microparticles, nanoparticles,lipid emulsions or similar formulations, which are especially necessaryfor inducing a strong response when the antigen is relatively weak. Theyalso accelerate the immune response and enable the antigen doses to bereduced by approximately two orders of magnitude, with comparableantibody responses to the full-dose vaccine without CpG in someexperiments (Krieg, 2006). U.S. Pat. No. 6,406,705 B1 describes thecombined use of CpG oligonucleotides, non-nucleic acid adjuvants and anantigen to induce an antigen-specific immune response. A CpG TLR9antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen(Berlin, Germany) which is a preferred component of the pharmaceuticalcomposition of the present invention. Other TLR binding molecules suchas RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.

Other examples for useful adjuvants include, but are not limited tochemically modified CpGs (e.g. CpR, Idera), dsRNA analogues such asPoly(I:C) and derivates thereof (e.g. AmpliGen®, Hiltonol®, poly-(ICLC),poly(IC—R), poly(I:C12U), non-CpG bacterial DNA or RNA as well asimmunoactive small molecules and antibodies such as cyclophosphamide,sunitinib, immune checkpoint inhibitors including ipilimumab, nivolumab,pembrolizumab, atezolizumab, avelumab, durvalumab, and cemiplimab,Bevacizumab®, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil,sorafenib, temozolomide, temsirolimus, XL-999, CP-547632, pazopanib,VEGF Trap, ZD2171, AZD2171, anti-CTLA4, other antibodies targeting keystructures of the immune system (e.g. anti-CD40, anti-TGFbeta,anti-TNFalpha receptor) and SC58175, which may act therapeuticallyand/or as an adjuvant. The amounts and concentrations of adjuvants andadditives useful in the context of the present invention can readily bedetermined by the skilled artisan without undue experimentation.

Preferred adjuvants are anti-CD40, imiquimod, resiquimod, GM-CSF,cyclophosphamide, sunitinib, atezolizumab, bevacizumab,interferon-alpha, interferon-beta, CpG oligonucleotides and derivatives,poly-(I:C) and derivatives, RNA, sildenafil, particulate formulationswith PLG, virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, IL-13,IL-15, IL-21, and IL-23.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimod,resiquimod, and interferon-alpha.

In a preferred embodiment, the pharmaceutical composition according tothe invention the adjuvant is selected from the group consisting ofcolony-stimulating factors, such as Granulocyte Macrophage ColonyStimulating Factor (GM-CSF, sargramostim), cyclophosphamide, imiquimodand resiquimod. In a preferred embodiment of the pharmaceuticalcomposition according to the invention, the adjuvant iscyclophosphamide, imiquimod or resiquimod. Even more preferred adjuvantsare Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, MontanideISA-51, poly-ICLC (Hiltonol®) and anti-CD40 mAB, or combinationsthereof.

This composition is used for parenteral administration, such assubcutaneous, intradermal, intramuscular or oral administration. Forthis, the peptides and optionally other molecules are dissolved orsuspended in a pharmaceutically acceptable, preferably aqueous carrier.In addition, the composition can contain excipients, such as buffers,binding agents, blasting agents, diluents, flavors, lubricants, etc. Thepeptides can also be administered together with immune stimulatingsubstances, such as cytokines. An extensive listing of excipients thatcan be used in such a composition, can be, for example, taken from A.Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). Thecomposition can be used for a prevention, prophylaxis and/or therapy ofadenomatous or cancerous diseases. Exemplary formulations can be foundin, for example, EP2112253.

It is important to realize that the immune response triggered by thevaccine according to the invention attacks the cancer in differentcell-stages and different stages of development. Furthermore, differentcancer associated signaling pathways are attacked. This is an advantageover vaccines that address only one or few targets, which may cause thetumor to easily adapt to the attack (tumor escape). Furthermore, not allindividual tumors express the same pattern of antigens. Therefore, acombination of several tumor-associated peptides ensures that everysingle tumor bears at least some of the targets. The composition isdesigned in such a way that each tumor is expected to express several ofthe antigens and cover several independent pathways necessary for tumorgrowth and maintenance. Thus, the vaccine can easily be used“off-the-shelf” for a larger patient population. This means that apre-selection of patients to be treated with the vaccine can berestricted to HLA typing, does not require any additional biomarkerassessments for antigen expression, but it is still ensured that severaltargets are simultaneously attacked by the induced immune response,which is important for efficacy (Banchereau et al., 2001; Walter et al.,2012).

As used herein, the term “scaffold” refers to a molecule thatspecifically binds to an (e.g. antigenic) determinant. In oneembodiment, a scaffold is able to direct the entity to which it isattached (e.g. a (second) antigen binding moiety) to a target site, forexample to a specific type of tumor cell or tumor stroma bearing theantigenic determinant (e.g. the complex of a peptide with MHC, accordingto the application at hand). In another embodiment a scaffold is able toactivate signaling through its target antigen, for example a T cellreceptor complex antigen. Scaffolds include but are not limited toantibodies and fragments thereof, antigen binding domains of anantibody, comprising an antibody heavy chain variable region and anantibody light chain variable region, binding proteins comprising atleast one ankyrin repeat motif and single domain antigen binding (SDAB)molecules, aptamers, (soluble) TCRs and (modified) cells such asallogenic or autologous T cells. To assess whether a molecule is ascaffold binding to a target, binding assays can be performed.

“Specific” binding means that the scaffold binds the peptide-MHC-complexof interest better than other naturally occurring peptide-MHC-complexes,to an extent that a scaffold armed with an active molecule that is ableto kill a cell bearing the specific target is not able to kill anothercell without the specific target but presenting other peptide-MHCcomplex(es). Binding to other peptide-MHC complexes is irrelevant if thepeptide of the cross-reactive peptide-MHC is not naturally occurring,i.e. not derived from the human HLA-peptidome. Tests to assess targetcell killing are well known in the art. They should be performed usingtarget cells (primary cells or cell lines) with unaltered peptide-MHCpresentation, or cells loaded with peptides such that naturallyoccurring peptide-MHC levels are reached.

Each scaffold can comprise a labelling which provides that the boundscaffold can be detected by determining the presence or absence of asignal provided by the label. For example, the scaffold can be labelledwith a fluorescent dye or any other applicable cellular marker molecule.Such marker molecules are well known in the art. For example, afluorescence-labelling, for example provided by a fluorescence dye, canprovide a visualization of the bound aptamer by fluorescence or laserscanning microscopy or flow cytometry.

Each scaffold can be conjugated with a second active molecule such asfor example IL-21, anti-CD3, and anti-CD28.

For further information on polypeptide scaffolds see for example thebackground section of WO 2014/071978A1 and the references cited therein.

The present invention further relates to aptamers. Aptamers (see forexample WO 2014/191359 and the literature as cited therein) are shortsingle-stranded nucleic acid molecules, which can fold into definedthree-dimensional structures and recognize specific target structures.They have appeared to be suitable alternatives for developing targetedtherapies. Aptamers have been shown to selectively bind to a variety ofcomplex targets with high affinity and specificity.

Aptamers recognizing cell surface located molecules have been identifiedwithin the past decade and provide means for developing diagnostic andtherapeutic approaches. Since aptamers have been shown to possess almostno toxicity and immunogenicity they are promising candidates forbiomedical applications. Indeed aptamers, for example prostate-specificmembrane-antigen recognizing aptamers, have been successfully employedfor targeted therapies and shown to be functional in xenograft in vivomodels. Furthermore, aptamers recognizing specific tumor cell lines havebeen identified.

DNA aptamers can be selected to reveal broad-spectrum recognitionproperties for various cancer cells, and particularly those derived fromsolid tumors, while non-tumorigenic and primary healthy cells are notrecognized. If the identified aptamers recognize not only a specifictumor sub-type but rather interact with a series of tumors, this rendersthe aptamers applicable as so-called broad-spectrum diagnostics andtherapeutics.

Further, investigation of cell-binding behavior with flow cytometryshowed that the aptamers revealed very good apparent affinities that arewithin the nanomolar range.

Aptamers are useful for diagnostic and therapeutic purposes. Further, itcould be shown that some of the aptamers are taken up by tumor cells andthus can function as molecular vehicles for the targeted delivery ofanti-cancer agents such as siRNA into tumor cells.

Aptamers can be selected against complex targets such as cells andtissues and complexes of the peptides comprising, preferably consistingof, a sequence according to any of SEQ ID NO 1 to SEQ ID NO 398,according to the invention at hand with the MHC molecule, using thecell-SELEX (Systematic Evolution of Ligands by Exponential enrichment)technique.

The peptides of the present invention can be used to generate anddevelop specific antibodies against MHC/peptide complexes. These can beused for therapy, targeting toxins or radioactive substances to thediseased tissue. Another use of these antibodies can be targetingradionuclides to the diseased tissue for imaging purposes such as PET.This use can help to detect small metastases or to determine the sizeand precise localization of diseased tissues.

Therefore, it is a further aspect of the invention to provide a methodfor producing a recombinant antibody specifically binding to a humanmajor histocompatibility complex (MHC) class I or II being complexedwith a HLA-restricted antigen (preferably a peptide according to thepresent invention), the method comprising: immunizing a geneticallyengineered non-human mammal comprising cells expressing said human majorhistocompatibility complex (MHC) class I or II with a soluble form of aMHC class I or II molecule being complexed with said HLA-restrictedantigen; isolating mRNA molecules from antibody producing cells of saidnon-human mammal; producing a phage display library displaying proteinmolecules encoded by said mRNA molecules; and isolating at least onephage from said phage display library, said at least one phagedisplaying said antibody specifically binding to said human majorhistocompatibility complex (MHC) class I or II being complexed with saidHLA-restricted antigen.

It is thus a further aspect of the invention to provide an antibody thatspecifically binds to a human major histocompatibility complex (MHC)class I or II being complexed with an HLA-restricted antigen, whereinthe antibody preferably is a polyclonal antibody, monoclonal antibody,bi-specific antibody and/or a chimeric antibody.

Respective methods for producing such antibodies and single chain classI major histocompatibility complexes, as well as other tools for theproduction of these antibodies are disclosed in WO 03/068201, WO2004/084798, WO 01/72768, WO 03/070752, and in publications (Cohen etal., 2003a; Cohen et al., 2003b; Denkberg et al., 2003), which for thepurposes of the present invention are all explicitly incorporated byreference in their entireties.

Preferably, the antibody is binding with a binding affinity of below 20nanomolar, preferably of below 10 nanomolar, to the complex, which isalso regarded as “specific” in the context of the present invention.

The present invention relates to a peptide comprising a sequence that isselected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 398, ora variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 398 or a variant thereof thatinduces T cells cross-reacting with said peptide, wherein said peptideis not the underlying full-length polypeptide.

The present invention further relates to a peptide comprising a sequencethat is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:398 or a variant thereof which is at least 88% homologous (preferablyidentical) to SEQ ID NO: 1 to SEQ ID NO: 398, wherein said peptide orvariant has an overall length of between 8 and 100, preferably between 8and 30, and most preferred between 8 and 14 amino acids.

The present invention further relates to the peptides according to theinvention that have the ability to bind to a molecule of the human majorhistocompatibility complex (MHC) class-I or -II.

The present invention further relates to the peptides according to theinvention wherein the peptide consists or consists essentially of anamino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 398.

The present invention further relates to the peptides according to theinvention, wherein the peptide is (chemically) modified and/or includesnon-peptide bonds.

The present invention further relates to the peptides according to theinvention, wherein the peptide is part of a fusion protein, inparticular comprising N-terminal amino acids of the HLA-DRantigen-associated invariant chain (Ii), or wherein the peptide is fusedto (or into) an antibody, such as, for example, an antibody that isspecific for dendritic cells.

The present invention further relates to a nucleic acid, encoding thepeptides according to the invention, provided that the peptide is notthe complete (full) human protein.

The present invention further relates to the nucleic acid according tothe invention that is DNA, cDNA, PNA, RNA or combinations thereof.

The present invention further relates to an expression vector capable ofexpressing a nucleic acid according to the present invention.

The present invention further relates to a peptide according to thepresent invention, a nucleic acid according to the present invention oran expression vector according to the present invention for use inmedicine, in particular in the treatment of acute myeloid leukemia,breast cancer, cholangiocellular carcinoma, chronic lymphocyticleukemia, colorectal cancer, gallbladder cancer, glioblastoma, gastriccancer, hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer.

The present invention further relates to a host cell comprising anucleic acid according to the invention or an expression vectoraccording to the invention.

The present invention further relates to the host cell according to thepresent invention that is an antigen presenting cell, and preferably adendritic cell.

The present invention further relates to a method of producing a peptideaccording to the present invention, said method comprising culturing thehost cell according to the present invention, and isolating the peptidefrom said host cell or its culture medium.

The present invention further relates to the method according to thepresent invention, where-in the antigen is loaded onto class I or II MHCmolecules expressed on the surface of a suitable antigen-presenting cellby contacting a sufficient amount of the antigen with anantigen-presenting cell.

The present invention further relates to the method according to theinvention, wherein the antigen-presenting cell comprises an expressionvector capable of expressing said peptide containing SEQ ID NO: 1 to SEQID NO: 398 or said variant amino acid sequence.

The present invention further relates to activated T cells, produced bythe method according to the present invention, wherein said T cellsselectively recognizes a cell which aberrantly expresses a polypeptidecomprising an amino acid sequence according to the present invention.

In an aspect, the activated T cells may be produced by contacting invitro T cells with antigen loaded human class I or II MHC moleculespresented on the surface of a suitable antigen-presenting cell or anartificial construct mimicking an antigen-presenting cell for a periodof time sufficient to activate said T cells.

The present invention further relates to a method of killing targetcells in a patient, in which the target cells aberrantly express apolypeptide comprising any amino acid sequence according to the presentinvention, the method comprising administering to the patient aneffective number of T cells as according to the present invention.

The present invention further relates to a method of treating a patientwho has cancer, in which the cancer cells aberrantly express apolypeptide comprising any amino acid sequence according to the presentinvention, the method comprising administering to the patient aneffective number of T cells as produced according to the presentinvention.

The present invention further relates to a method of eliciting an immuneresponse in a patient who has cancer, in which the cancer cellsaberrantly express a polypeptide comprising any amino acid sequenceaccording to the present invention, the method comprising administeringto the patient an effective number of T cells as produced according tothe present invention.

In an aspect, the T cells may be autologous to the patient. In anotheraspect, the T cells may be obtained from a healthy donor. In anotheraspect, the T cells may be derived from tumor infiltrating lymphocytesor peripheral blood mononuclear cells. In another aspect, the immuneresponse may include cytotoxic T cell response.

The present invention further relates to the use of any peptidedescribed, a nucleic acid according to the present invention, anexpression vector according to the present invention, a cell accordingto the present invention, or an activated cytotoxic T lymphocyteaccording to the present invention as a medicament or in the manufactureof a medicament. The present invention further relates to a useaccording to the present invention, wherein the medicament is activeagainst cancer.

The present invention further relates to a use according to theinvention, wherein the medicament is a vaccine. The present inventionfurther relates to a use according to the invention, wherein themedicament is active against cancer.

The present invention further relates to a use according to theinvention, wherein said cancer cells are acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer cells or other solidor hematological tumor cells such as acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer.

The present invention further relates to particular marker proteins andbiomarkers based on the peptides according to the present invention,herein called “targets” that can be used in the diagnosis and/orprognosis of acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lungcancer (including non-small cell lung cancer adenocarcinoma, squamouscell non-small cell lung cancer, and small cell lung cancer), ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, urinary bladder carcinoma, uterine and endometrialcancer. The present invention also relates to the use of these noveltargets for cancer treatment.

The term “antibody” or “antibodies” is used herein in a broad sense andincludes both polyclonal and monoclonal antibodies. In addition tointact or “full” immunoglobulin molecules, also included in the term“antibodies” are fragments (e.g. CDRs, Fv, Fab and Fc fragments) orpolymers of those immunoglobulin molecules and humanized versions ofimmunoglobulin molecules, as long as they exhibit any of the desiredproperties (e.g., specific binding of an acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer marker (poly)peptide,delivery of a toxin to an acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer cell expressing a cancer marker gene atan increased level, and/or inhibiting the activity of an acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancermarker polypeptide) according to the invention.

Whenever possible, the antibodies of the invention may be purchased fromcommercial sources. The antibodies of the invention may also begenerated using well-known methods. The skilled artisan will understandthat either full length acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer marker polypeptides or fragments thereofmay be used to generate the antibodies of the invention. A polypeptideto be used for generating an antibody of the invention may be partiallyor fully purified from a natural source or may be produced usingrecombinant DNA techniques.

For example, a cDNA encoding a peptide according to the presentinvention, such as a peptide according to SEQ ID NO: 1 to SEQ ID NO: 398polypeptide, or a variant or fragment thereof, can be expressed inprokaryotic cells (e.g., bacteria) or eukaryotic cells (e.g., yeast,insect, or mammalian cells), after which the recombinant protein can bepurified and used to generate a monoclonal or polyclonal antibodypreparation that specifically bind the acute myeloid leukemia, breastcancer, cholangiocellular carcinoma, chronic lymphocytic leukemia,colorectal cancer, gallbladder cancer, glioblastoma, gastric cancer,hepatocellular carcinoma, head and neck squamous cell carcinoma,melanoma, non-Hodgkin lymphoma, lung cancer (including non-small celllung cancer adenocarcinoma, squamous cell non-small cell lung cancer,and small cell lung cancer), ovarian cancer, esophageal cancer,pancreatic cancer, prostate cancer, renal cell carcinoma, urinarybladder carcinoma, uterine and endometrial cancer marker polypeptideused to generate the antibody according to the invention.

One of skill in the art will realize that the generation of two or moredifferent sets of monoclonal or polyclonal antibodies maximizes thelikelihood of obtaining an antibody with the specificity and affinityrequired for its intended use (e.g., ELISA, immunohistochemistry, invivo imaging, immunotoxin therapy). The antibodies are tested for theirdesired activity by known methods, in accordance with the purpose forwhich the antibodies are to be used (e.g., ELISA, immunohistochemistry,immunotherapy, etc.; for further guidance on the generation and testingof antibodies, see, e.g., Greenfield, 2014 (Greenfield, 2014)). Forexample, the antibodies may be tested in ELISA assays or, Western blots,immunohistochemical staining of formalin-fixed cancers or frozen tissuesections. After their initial in vitro characterization, antibodiesintended for therapeutic or in vivo diagnostic use are tested accordingto known clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e.; the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. The monoclonal antibodies herein specifically include“chimeric” antibodies in which a portion of the heavy and/or light chainis identical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired antagonistic activity (U.S. Pat. No. 4,816,567, which is herebyincorporated in its entirety).

Monoclonal antibodies of the invention may be prepared using hybridomamethods. In a hybridoma method, a mouse or other appropriate host animalis typically immunized with an immunizing agent to elicit lymphocytesthat produce or are capable of producing antibodies that willspecifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies).

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 and U.S. Pat. No.4,342,566. Papain digestion of antibodies typically produces twoidentical antigen binding fragments, called Fab fragments, each with asingle antigen binding site, and a residual Fc fragment. Pepsintreatment yields a F(ab′)2 fragment and a pFc′ fragment.

The antibody fragments, whether attached to other sequences or not, canalso include insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the fragment is not significantly altered orimpaired compared to the non-modified antibody or antibody fragment.These modifications can provide for some additional property, such as toremove/add amino acids capable of disulfide bonding, to increase itsbio-longevity, to alter its secretory characteristics, etc. In any case,the antibody fragment must possess a bioactive property, such as bindingactivity, regulation of binding at the binding domain, etc. Functionalor active regions of the antibody may be identified by mutagenesis of aspecific region of the protein, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the antibody fragment.

The antibodies of the invention may further comprise humanizedantibodies or human antibodies. Humanized forms of non-human (e.g.,murine) antibodies are chimeric immunoglobulins, immunoglobulin chainsor fragments thereof (such as Fv, Fab, Fab′ or other antigen-bindingsubsequences of antibodies) which contain minimal sequence derived fromnon-human immunoglobulin. Humanized antibodies include humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat or rabbit having the desired specificity, affinity andcapacity. In some instances, Fv framework (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin.

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed by substituting rodent CDRs or CDR sequencesfor the corresponding sequences of a human antibody. Accordingly, such“humanized” antibodies are chimeric antibodies (U.S. Pat. No.4,816,567), wherein substantially less than an intact human variabledomain has been substituted by the corresponding sequence from anon-human species. In practice, humanized antibodies are typically humanantibodies in which some CDR residues and possibly some FR residues aresubstituted by residues from analogous sites in rodent antibodies.

Transgenic animals (e.g., mice) that are capable, upon immunization, ofproducing a full repertoire of human antibodies in the absence ofendogenous immunoglobulin production can be employed. For example, ithas been described that the homozygous deletion of the antibody heavychain joining region gene in chimeric and germ-line mutant mice resultsin complete inhibition of endogenous antibody production. Transfer ofthe human germ-line immunoglobulin gene array in such germ-line mutantmice will result in the production of human antibodies upon antigenchallenge. Human antibodies can also be produced in phage displaylibraries.

Antibodies of the invention are preferably administered to a subject ina pharmaceutically acceptable carrier. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include saline, Ringer's solutionand dextrose solution. The pH of the solution is preferably from about 5to about 8, and more preferably from about 7 to about 7.5. Furthercarriers include sustained release preparations such as semipermeablematrices of solid hydrophobic polymers containing the antibody, whichmatrices are in the form of shaped articles, e.g., films, liposomes ormicroparticles. It will be apparent to those persons skilled in the artthat certain carriers may be more preferable depending upon, forinstance, the route of administration and concentration of antibodybeing administered.

The antibodies can be administered to the subject, patient, or cell byinjection (e.g., intravenous, intraperitoneal, subcutaneous,intramuscular), or by other methods such as infusion that ensure itsdelivery to the bloodstream in an effective form. The antibodies mayalso be administered by intratumoral or peritumoral routes, to exertlocal as well as systemic therapeutic effects. Local or intravenousinjection is preferred.

Effective dosages and schedules for administering the antibodies may bedetermined empirically, and making such determinations is within theskill in the art. Those skilled in the art will understand that thedosage of antibodies that must be administered will vary depending on,for example, the subject that will receive the antibody, the route ofadministration, the particular type of antibody used and other drugsbeing administered. A typical daily dosage of the antibody used alonemight range from about 1 (μg/kg to up to 100 mg/kg of body weight ormore per day, depending on the factors mentioned above. Followingadministration of an antibody, preferably for treating acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer,the efficacy of the therapeutic antibody can be assessed in various wayswell known to the skilled practitioner. For instance, the size, number,and/or distribution of cancer in a subject receiving treatment may bemonitored using standard tumor imaging techniques. Atherapeutically-administered antibody that arrests tumor growth, resultsin tumor shrinkage, and/or prevents the development of new tumors,compared to the disease course that would occurs in the absence ofantibody administration, is an efficacious antibody for treatment ofcancer.

It is a further aspect of the invention to provide a method forproducing a soluble T-cell receptor (sTCR) recognizing a specificpeptide-MHC complex. Such soluble T-cell receptors can be generated fromspecific T-cell clones, and their affinity can be increased bymutagenesis targeting the complementarity-determining regions. For thepurpose of T-cell receptor selection, phage display can be used (US2010/0113300, (Liddy et al., 2012)). For the purpose of stabilization ofT-cell receptors during phage display and in case of practical use asdrug, alpha and beta chain can be linked e.g. by non-native disulfidebonds, other covalent bonds (single-chain T-cell receptor), or bydimerization domains (Boulter et al., 2003; Card et al., 2004; Willcoxet al., 1999). The T-cell receptor can be linked to toxins, drugs,cytokines (see, for example, US 2013/0115191), and domains recruitingeffector cells such as an anti-CD3 domain, etc., in order to executeparticular functions on target cells. Moreover, it could be expressed inT cells used for adoptive transfer. Further information can be found inWO 2004/033685A1 and WO 2004/074322A1. A combination of sTCRs isdescribed in WO 2012/056407A1. Further methods for the production aredisclosed in WO 2013/057586A1.

In addition, the peptides and/or the TCRs or antibodies or other bindingmolecules of the present invention can be used to verify a pathologist'sdiagnosis of a cancer based on a biopsied sample.

The antibodies or TCRs may also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radionucleotide (such as¹¹¹In, ⁹⁹Tc, ¹⁴C, ¹³¹I, ³H, ³²P or ³⁵S) so that the tumor can belocalized using immunoscintiography.

In one embodiment, antibodies or fragments thereof bind to theextracellular domains of two or more targets of a protein selected fromthe group consisting of the above-mentioned proteins, and the affinityvalue (Kd) is less than 1×10 μM.

Antibodies for diagnostic use may be labeled with probes suitable fordetection by various imaging methods. Methods for detection of probesinclude, but are not limited to, fluorescence, light, confocal andelectron microscopy; magnetic resonance imaging and spectroscopy;fluoroscopy, computed tomography and positron emission tomography.Suitable probes include, but are not limited to, fluorescein, rhodamine,eosin and other fluorophores, radioisotopes, gold, gadolinium and otherlanthanides, paramagnetic iron, fluorine-18 and other positron-emittingradionuclides. Additionally, probes may be bi- or multi-functional andbe detectable by more than one of the methods listed. These antibodiesmay be directly or indirectly labeled with said probes. Attachment ofprobes to the antibodies includes covalent attachment of the probe,incorporation of the probe into the antibody, and the covalentattachment of a chelating compound for binding of probe, amongst otherswell recognized in the art. For immunohistochemistry, the disease tissuesample may be fresh or frozen or may be embedded in paraffin and fixedwith a preservative such as formalin. The fixed or embedded sectioncontains the sample are contacted with a labeled primary antibody andsecondary antibody, wherein the antibody is used to detect theexpression of the proteins in situ.

Another aspect of the present invention includes an in vitro method forproducing activated T cells, the method comprising contacting in vitro Tcells with antigen loaded human MHC molecules expressed on the surfaceof a suitable antigen-presenting cell for a period of time sufficient toactivate the T cell in an antigen specific manner, wherein the antigenis a peptide according to the invention. Preferably a sufficient amountof the antigen is used with an antigen-presenting cell.

Preferably the mammalian cell lacks or has a reduced level or functionof the TAP peptide transporter. Suitable cells that lack the TAP peptidetransporter include T2, RMA-S and Drosophila cells. TAP is thetransporter associated with antigen processing.

The human peptide loading deficient cell line T2 is available from theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.20852, USA under Catalogue No CRL 1992; the Drosophila cell lineSchneider line 2 is available from the ATCC under Catalogue No CRL19863; the mouse RMA-S cell line is described in Ljunggren et al.(Ljunggren and Karre, 1985).

Preferably, before transfection the host cell expresses substantially noMHC class I molecules. It is also preferred that the stimulator cellexpresses a molecule important for providing a co-stimulatory signal forT-cells such as any of B7.1, B7.2, ICAM-1 and LFA 3. The nucleic acidsequences of numerous MHC class I molecules and of the co-stimulatormolecules are publicly available from the GenBank and EMBL databases.

In case of an MHC class I epitope being used as an antigen; the T cellsare CD8-positive T cells.

If an antigen-presenting cell is transfected to express such an epitope,preferably the cell comprises an expression vector capable of expressinga peptide containing SEQ ID NO: 1 to SEQ ID NO: 398, or a variant aminoacid sequence thereof.

A number of other methods may be used for generating T cells in vitro.For example, autologous tumor-infiltrating lymphocytes can be used inthe generation of CTL. Plebanski et al. (Plebanski et al., 1995) madeuse of autologous peripheral blood lymphocytes (PLBs) in the preparationof T cells. Furthermore, the production of autologous T cells by pulsingdendritic cells with peptide or polypeptide, or via infection withrecombinant virus is possible. Also, B cells can be used in theproduction of autologous T cells. In addition, macrophages pulsed withpeptide or polypeptide, or infected with recombinant virus, may be usedin the preparation of autologous T cells. S. Walter et al. (Walter etal., 2003) describe the in vitro priming of T cells by using artificialantigen presenting cells (aAPCs), which is also a suitable way forgenerating T cells against the peptide of choice. In the presentinvention, aAPCs were generated by the coupling of preformed MHC:peptidecomplexes to the surface of polystyrene particles (microbeads) bybiotin:streptavidin biochemistry. This system permits the exact controlof the MHC density on aAPCs, which allows to selectively elicit high- orlow-avidity antigen-specific T cell responses with high efficiency fromblood samples. Apart from MHC:peptide complexes, aAPCs should carryother proteins with co-stimulatory activity like anti-CD28 antibodiescoupled to their surface. Furthermore, such aAPC-based systems oftenrequire the addition of appropriate soluble factors, e. g. cytokines,like interleukin-12.

Allogeneic cells may also be used in the preparation of T cells and amethod is described in detail in WO 97/26328, incorporated herein byreference. For example, in addition to Drosophila cells and T2 cells,other cells may be used to present antigens such as CHO cells,baculovirus-infected insect cells, bacteria, yeast, andvaccinia-infected target cells. In addition plant viruses may be used(see, for example, Porta et al. (Porta et al., 1994) which describes thedevelopment of cowpea mosaic virus as a high-yielding system for thepresentation of foreign peptides.

The activated T cells that are directed against the peptides of theinvention are useful in therapy. Thus, a further aspect of the inventionprovides activated T cells obtainable by the foregoing methods of theinvention.

Activated T cells, which are produced by the above method, willselectively recognize a cell that aberrantly expresses a polypeptidethat comprises an amino acid sequence of SEQ ID NO: 1 to SEQ ID NO 398.

Preferably, the T cell recognizes the cell by interacting through itsTCR with the HLA/peptide-complex (for example, binding). The T cells areuseful in a method of killing target cells in a patient whose targetcells aberrantly express a polypeptide comprising an amino acid sequenceof the invention wherein the patient is administered an effective numberof the activated T cells. The T cells that are administered to thepatient may be derived from the patient and activated as described above(i.e. they are autologous T cells). Alternatively, the T cells are notfrom the patient but are from another individual. Of course, it ispreferred if the individual is a healthy individual. By “healthyindividual” the inventors mean that the individual is generally in goodhealth, preferably has a competent immune system and, more preferably,is not suffering from any disease that can be readily tested for anddetected.

In vivo, the target cells for the CD8-positive T cells according to thepresent invention can be cells of the tumor (which sometimes express MHCclass II) and/or stromal cells surrounding the tumor (tumor cells)(which sometimes also express MHC class II; (Dengjel et al., 2006)).

The T cells of the present invention may be used as active ingredientsof a therapeutic composition. Thus, the invention also provides a methodof killing target cells in a patient whose target cells aberrantlyexpress a polypeptide comprising an amino acid sequence of theinvention, the method comprising administering to the patient aneffective number of T cells as defined above.

By “aberrantly expressed” the inventors also mean that the polypeptideis over-expressed compared to levels of expression in normal tissues orthat the gene is silent in the tissue from which the tumor is derivedbut, in the tumor, it is expressed. By “over-expressed” the inventorsmean that the polypeptide is present at a level at least 1.2-fold ofthat present in normal tissue; preferably at least 2-fold, and morepreferably at least 5-fold or 10-fold the level present in normaltissue.

T cells may be obtained by methods known in the art, e.g. thosedescribed above.

Protocols for this so-called adoptive transfer of T cells are well knownin the art. Reviews can be found in: Gattioni et al. and Morgan et al.(Gattinoni et al., 2006; Morgan et al., 2006).

Another aspect of the present invention includes the use of the peptidescomplexed with MHC to generate a T-cell receptor whose nucleic acid iscloned and is introduced into a host cell, preferably a T cell. Thisengineered T cell can then be transferred to a patient for therapy ofcancer.

Any molecule of the invention, i.e. the peptide, nucleic acid, antibody,expression vector, cell, activated T cell, T-cell receptor or thenucleic acid encoding it, is useful for the treatment of disorders,characterized by cells escaping an immune response. Therefore, anymolecule of the present invention may be used as medicament or in themanufacture of a medicament. The molecule may be used by itself orcombined with other molecule(s) of the invention or (a) knownmolecule(s).

The present invention is further directed at a kit comprising:

(a) a container containing a pharmaceutical composition as describedabove, in solution or in lyophilized form;(b) optionally a second container containing a diluent or reconstitutingsolution for the lyophilized formulation; and(c) optionally, instructions for (i) use of the solution or (ii)reconstitution and/or use of the lyophilized formulation.

The kit may further comprise one or more of (iii) a buffer, (iv) adiluent, (v) a filter, (vi) a needle, or (v) a syringe. The container ispreferably a bottle, a vial, a syringe or test tube; and it may be amulti-use container. The pharmaceutical composition is preferablylyophilized.

Kits of the present invention preferably comprise a lyophilizedformulation of the present invention in a suitable container andinstructions for its reconstitution and/or use. Suitable containersinclude, for example, bottles, vials (e.g. dual chamber vials), syringes(such as dual chamber syringes) and test tubes. The container may beformed from a variety of materials such as glass or plastic. Preferablythe kit and/or container contain/s instructions on or associated withthe container that indicates directions for reconstitution and/or use.For example, the label may indicate that the lyophilized formulation isto be reconstituted to peptide concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor subcutaneous administration.

The container holding the formulation may be a multi-use vial, whichallows for repeat administrations (e.g., from 2-6 administrations) ofthe reconstituted formulation. The kit may further comprise a secondcontainer comprising a suitable diluent (e.g., sodium bicarbonatesolution).

Upon mixing of the diluent and the lyophilized formulation, the finalpeptide concentration in the reconstituted formulation is preferably atleast 0.15 mg/mL/peptide (=75 μg) and preferably not more than 3mg/mL/peptide (=1500 μg). The kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use.

Kits of the present invention may have a single container that containsthe formulation of the pharmaceutical compositions according to thepresent invention with or without other components (e.g., othercompounds or pharmaceutical compositions of these other compounds) ormay have distinct container for each component.

Preferably, kits of the invention include a formulation of the inventionpackaged for use in combination with the co-administration of a secondcompound (such as adjuvants (e.g. GM-CSF), a chemotherapeutic agent, anatural product, a hormone or antagonist, an anti-angiogenesis agent orinhibitor, an apoptosis-inducing agent or a chelator) or apharmaceutical composition thereof. The components of the kit may bepre-complexed or each component may be in a separate distinct containerprior to administration to a patient. The components of the kit may beprovided in one or more liquid solutions, preferably, an aqueoussolution, more preferably, a sterile aqueous solution. The components ofthe kit may also be provided as solids, which may be converted intoliquids by addition of suitable solvents, which are preferably providedin another distinct container.

The container of a therapeutic kit may be a vial, test tube, flask,bottle, syringe, or any other means of enclosing a solid or liquid.Usually, when there is more than one component, the kit will contain asecond vial or other container, which allows for separate dosing. Thekit may also contain another container for a pharmaceutically acceptableliquid. Preferably, a therapeutic kit will contain an apparatus (e.g.,one or more needles, syringes, eye droppers, pipette, etc.), whichenables administration of the agents of the invention that arecomponents of the present kit.

The present formulation is one that is suitable for administration ofthe peptides by any acceptable route such as oral (enteral), nasal,ophthal, subcutaneous, intradermal, intramuscular, intravenous ortransdermal. Preferably, the administration is s.c., and most preferablyi.d. administration may be by infusion pump.

Since the peptides of the invention were isolated from acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer,the medicament of the invention is preferably used to treat acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer.

The present invention further relates to a method for producing apersonalized pharmaceutical for an individual patient comprisingmanufacturing a pharmaceutical composition comprising at least onepeptide selected from a warehouse of pre-screened TUMAPs, wherein the atleast one peptide used in the pharmaceutical composition is selected forsuitability in the individual patient. In one embodiment, thepharmaceutical composition is a vaccine. The method could also beadapted to produce T cell clones for down-stream applications, such asTCR isolations, or soluble antibodies, and other treatment options.

A “personalized pharmaceutical” shall mean specifically tailoredtherapies for one individual patient that will only be used for therapyin such individual patient, including actively personalized cancervaccines and adoptive cellular therapies using autologous patienttissue.

As used herein, the term “warehouse” shall refer to a group or set ofpeptides that have been pre-screened for immunogenicity and/orover-presentation in a particular tumor type. The term “warehouse” isnot intended to imply that the particular peptides included in thevaccine have been pre-manufactured and stored in a physical facility,although that possibility is contemplated. It is expressly contemplatedthat the peptides may be manufactured de novo for each individualizedvaccine produced or may be pre-manufactured and stored. The warehouse(e.g. in the form of a database) is composed of tumor-associatedpeptides which were highly overexpressed in the tumor tissue of acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancerpatients with various HLA-A HLA-B and HLA-C alleles. It may contain MHCclass I and MHC class II peptides or elongated MHC class I peptides. Inaddition to the tumor associated peptides collected from several acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancertissues, the warehouse may contain HLA-A*02, HLA-A*01, HLA-A*03,HLA-A*24, HLA-B*07, HLA-B*08 and HLA-B*44 marker peptides. Thesepeptides allow comparison of the magnitude of T-cell immunity induced byTUMAPS in a quantitative manner and hence allow important conclusion tobe drawn on the capacity of the vaccine to elicit anti-tumor responses.Secondly, they function as important positive control peptides derivedfrom a “non-self” antigen in the case that any vaccine-induced T-cellresponses to TUMAPs derived from “self” antigens in a patient are notobserved. And thirdly, it may allow conclusions to be drawn, regardingthe status of immunocompetence of the patient. TUMAPs for the warehouseare identified by using an integrated functional genomics approachcombining gene expression analysis, mass spectrometry, and T-cellimmunology (XPresident®). The approach assures that only TUMAPs trulypresent on a high percentage of tumors but not or only minimallyexpressed on normal tissue, are chosen for further analysis. For initialpeptide selection, acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer samples from patients and blood fromhealthy donors were analyzed in a stepwise approach:

1. HLA ligands from the malignant material were identified by massspectrometry2. Genome-wide messenger ribonucleic acid (mRNA) expression analysis wasused to identify genes over-expressed in the malignant tissue (acutemyeloid leukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer)compared with a range of normal organs and tissues3. Identified HLA ligands were compared to gene expression data.Peptides over-presented or selectively presented on tumor tissue,preferably encoded by selectively expressed or over-expressed genes asdetected in step 2 were considered suitable TUMAP candidates for amulti-peptide vaccine.4. Literature research was performed in order to identify additionalevidence supporting the relevance of the identified peptides as TUMAPs5. The relevance of over-expression at the mRNA level was confirmed byredetection of selected TUMAPs from step 3 on tumor tissue and lack of(or infrequent) detection on healthy tissues.6. In order to assess, whether an induction of in vivo T-cell responsesby the selected peptides may be feasible, in vitro immunogenicity assayswere performed using human T cells from healthy donors as well as fromacute myeloid leukemia, breast cancer, cholangiocellular carcinoma,chronic lymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancerpatients.

In an aspect, the peptides are pre-screened for immunogenicity beforebeing included in the warehouse. By way of example, and not limitation,the immunogenicity of the peptides included in the warehouse isdetermined by a method comprising in vitro T-cell priming throughrepeated stimulations of CD8+ T cells from healthy donors withartificial antigen presenting cells loaded with peptide/MHC complexesand anti-CD28 antibody.

This method is preferred for rare cancers and patients with a rareexpression profile. In contrast to multi-peptide cocktails with a fixedcomposition as currently developed, the warehouse allows a significantlyhigher matching of the actual expression of antigens in the tumor withthe vaccine. Selected single or combinations of several “off-the-shelf”peptides will be used for each patient in a multitarget approach. Intheory an approach based on selection of e.g. 5 different antigenicpeptides from a library of 50 would already lead to approximately 17million possible drug product (DP) compositions.

In an aspect, the peptides are selected for inclusion in the vaccinebased on their suitability for the individual patient based on themethod according to the present invention as described herein, or asbelow.

The HLA phenotype, transcriptomic and peptidomic data is gathered fromthe patient's tumor material, and blood samples to identify the mostsuitable peptides for each patient containing “warehouse” andpatient-unique (i.e. mutated) TUMAPs. Those peptides will be chosen,which are selectively or over-expressed in the patient's tumor and,where possible, show strong in vitro immunogenicity if tested with thepatients' individual PBMCs.

Preferably, the peptides included in the vaccine are identified by amethod comprising: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; (b) comparingthe peptides identified in (a) with a warehouse (database) of peptidesas described above; and (c) selecting at least one peptide from thewarehouse (database) that correlates with a tumor-associated peptideidentified in the patient. For example, the TUMAPs presented by thetumor sample are identified by: (a1) comparing expression data from thetumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. Preferably, the sequences of MHCligands are identified by eluting bound peptides from MHC moleculesisolated from the tumor sample and sequencing the eluted ligands.Preferably, the tumor sample and the normal tissue are obtained from thesame patient.

In addition to, or as an alternative to, selecting peptides using awarehousing (database) model, TUMAPs may be identified in the patient denovo, and then included in the vaccine. As one example, candidate TUMAPsmay be identified in the patient by (a1) comparing expression data fromthe tumor sample to expression data from a sample of normal tissuecorresponding to the tissue type of the tumor sample to identifyproteins that are over-expressed or aberrantly expressed in the tumorsample; and (a2) correlating the expression data with sequences of MHCligands bound to MHC class I and/or class II molecules in the tumorsample to identify MHC ligands derived from proteins over-expressed oraberrantly expressed by the tumor. As another example, proteins may beidentified containing mutations that are unique to the tumor samplerelative to normal corresponding tissue from the individual patient, andTUMAPs can be identified that specifically target the mutation. Forexample, the genome of the tumor and of corresponding normal tissue canbe sequenced by whole genome sequencing: For discovery of non-synonymousmutations in the protein-coding regions of genes, genomic DNA and RNAare extracted from tumor tissues and normal non-mutated genomic germlineDNA is extracted from peripheral blood mononuclear cells (PBMCs). Theapplied NGS approach is confined to the re-sequencing of protein codingregions (exome re-sequencing). For this purpose, exonic DNA from humansamples is captured using vendor-supplied target enrichment kits,followed by sequencing with e.g. a HiSeq2000 (Illumina). Additionally,tumor mRNA is sequenced for direct quantification of gene expression andvalidation that mutated genes are expressed in the patients' tumors. Theresultant millions of sequence reads are processed through softwarealgorithms. The output list contains mutations and gene expression.Tumor-specific somatic mutations are determined by comparison with thePBMC-derived germline variations and prioritized. The de novo identifiedpeptides can then be tested for immunogenicity as described above forthe warehouse, and candidate TUMAPs possessing suitable immunogenicityare selected for inclusion in the vaccine.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient by the method asdescribed above; (b) comparing the peptides identified in a) with awarehouse of peptides that have been prescreened for immunogenicity andoverpresentation in tumors as compared to corresponding normal tissue;(c) selecting at least one peptide from the warehouse that correlateswith a tumor-associated peptide identified in the patient; and (d)optionally, selecting at least one peptide identified de novo in (a)confirming its immunogenicity.

In one exemplary embodiment, the peptides included in the vaccine areidentified by: (a) identifying tumor-associated peptides (TUMAPs)presented by a tumor sample from the individual patient; and (b)selecting at least one peptide identified de novo in (a) and confirmingits immunogenicity.

Once the peptides for a personalized peptide based vaccine are selected,the vaccine is produced. The vaccine preferably is a liquid formulationconsisting of the individual peptides dissolved in between 20-40% DMSO,preferably about 30-35% DMSO, such as about 33% DMSO.

Each peptide to be included into a product is dissolved in DMSO. Theconcentration of the single peptide solutions has to be chosen dependingon the number of peptides to be included into the product. The singlepeptide-DMSO solutions are mixed in equal parts to achieve a solutioncontaining all peptides to be included in the product with aconcentration of ˜2.5 mg/ml per peptide. The mixed solution is thendiluted 1:3 with water for injection to achieve a concentration of 0.826mg/ml per peptide in 33% DMSO. The diluted solution is filtered througha 0.22 μm sterile filter. The final bulk solution is obtained.

Final bulk solution is filled into vials and stored at −20° C. untiluse. One vial contains 700 μL solution, containing 0.578 mg of eachpeptide. Of this, 500 μL (approx. 400 μg per peptide) will be appliedfor intradermal injection.

In addition to being useful for treating cancer, the peptides of thepresent invention are also useful as diagnostics. Since the peptideswere generated from acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer cells and since it was determined thatthese peptides are not or at lower levels present in normal tissues,these peptides can be used to diagnose the presence of a cancer.

The presence of claimed peptides on tissue biopsies in blood samples canassist a pathologist in diagnosis of cancer. Detection of certainpeptides by means of antibodies, mass spectrometry or other methodsknown in the art can tell the pathologist that the tissue sample ismalignant or inflamed or generally diseased, or can be used as abiomarker for acute myeloid leukemia, breast cancer, cholangiocellularcarcinoma, chronic lymphocytic leukemia, colorectal cancer, gallbladdercancer, glioblastoma, gastric cancer, hepatocellular carcinoma, head andneck squamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lungcancer (including non-small cell lung cancer adenocarcinoma, squamouscell non-small cell lung cancer, and small cell lung cancer), ovariancancer, esophageal cancer, pancreatic cancer, prostate cancer, renalcell carcinoma, urinary bladder carcinoma, uterine and endometrialcancer. Presence of groups of peptides can enable classification orsub-classification of diseased tissues.

The detection of peptides on diseased tissue specimen can enable thedecision about the benefit of therapies involving the immune system,especially if T-lymphocytes are known or expected to be involved in themechanism of action. Loss of MHC expression is a well describedmechanism by which infected of malignant cells escapeimmuno-surveillance. Thus, presence of peptides shows that thismechanism is not exploited by the analyzed cells.

The peptides of the present invention might be used to analyzelymphocyte responses against those peptides such as T cell responses orantibody responses against the peptide or the peptide complexed to MHCmolecules. These lymphocyte responses can be used as prognostic markersfor decision on further therapy steps. These responses can also be usedas surrogate response markers in immunotherapy approaches aiming toinduce lymphocyte responses by different means, e.g. vaccination ofprotein, nucleic acids, autologous materials, adoptive transfer oflymphocytes. In gene therapy settings, lymphocyte responses againstpeptides can be considered in the assessment of side effects. Monitoringof lymphocyte responses might also be a valuable tool for follow-upexaminations of transplantation therapies, e.g. for the detection ofgraft versus host and host versus graft diseases.

The present invention will now be described in the following exampleswhich describe preferred embodiments thereof, and with reference to theaccompanying figures, nevertheless, without being limited thereto. Forthe purposes of the present invention, all references as cited hereinare incorporated by reference in their entireties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A through 1N show the over-presentation of various peptides indifferent cancer tissues (black dots). Upper part: Median MS signalintensities from technical replicate measurements are plotted as dotsfor single HLA-A*01 positive normal (grey dots, left part of figure) andtumor samples (black dots, right part of figure) on which the peptidewas detected. Boxes display median, 25th and 75th percentile ofnormalized signal intensities, while whiskers extend to the lowest datapoint still within 1.5 interquartile range (IQR) of the lower quartile,and the highest data point still within 1.5 IQR of the upper quartile.Normal organs are ordered according to risk categories (blood cells,blood vessels, brain, liver, lung: high risk, grey dots; reproductiveorgans, breast, prostate: low risk, grey dots; all other organs: mediumrisk; grey dots). Lower part: The relative peptide detection frequencyin every organ is shown as spine plot. Numbers below the panel indicatenumber of samples on which the peptide was detected out of the totalnumber of samples analyzed for each organ (N=72 for normal samples,N=155 for tumor samples). If the peptide has been detected on a samplebut could not be quantified for technical reasons, the sample isincluded in this representation of detection frequency, but no dot isshown in the upper part of the figure. Tissues (from left to right):Normal samples: blood cells; bloodvess (blood vessels); brain; heart;liver; lung; bile duct; bladder; bone marrow; esoph (esophagus); intest.la (large intestine); kidney; nerve periph (peripheral nerve); skin;spinal cord; spleen; stomach; thyroid; trachea. Tumor samples: AML(acute myeloid leukemia); BRCA (breast cancer); CCC (cholangiocellularcarcinoma); CLL (chronic lymphocytic leukemia); CRC (colorectal cancer);GBC (gallbladder cancer); GBM (glioblastoma); GC (gastric cancer); GEJC(gastro-esophageal junction cancer); HCC (hepatocellular carcinoma);HNSCC (head and neck squamous cell carcinoma); MEL (melanoma); NHL(non-Hodgkin lymphoma); NSCLCadeno (non-small cell lung canceradenocarcinoma); NSCLCother (NSCLC samples that could not unambiguouslybe assigned to NSCLCadeno or NSCLCsquam); NSCLCsquam (squamous cellnon-small cell lung cancer); OC (ovarian cancer); OSCAR (esophagealcancer); PACA (pancreatic cancer); PRCA (prostate cancer); RCC (renalcell carcinoma); SCLC (small cell lung cancer); UBC (urinary bladdercarcinoma); UEC (uterine and endometrial cancer).

FIG. 1A) Peptide: NSDISIPEY (SEQ ID NO.: 16), FIG. 1B) Peptide:TSDQLGYSY (SEQ ID NO.: 40), FIG. 1C) Peptide: HSDLLEDSKY (SEQ ID NO.:41), FIG. 1D) Peptide: SSDFDPLVY (SEQ ID NO.: 65), FIG. 1E) Peptide:YTELVEEKY (SEQ ID NO.: 70), FIG. 1F) Peptide: QTDVERIKDTY (SEQ ID NO.:76), FIG. 1G) Peptide: QLDSAVKNLY (SEQ ID NO.: 78), FIG. 1H) Peptide:HMLAAMAY (SEQ ID NO.: 296), FIG. 1I) Peptide: YTCEECGQAF (SEQ ID NO.:316), FIG. 1J) Peptide: NTDSMTLNNTAY (SEQ ID NO.: 329), FIG. 1K)Peptide: TLDSTRTLY (SEQ ID NO.: 1), FIG. 1L) Peptide: YLDSSKPAVY (SEQ IDNO.: 15), FIG. 1M) Peptide: PSEVPVDSHYY (SEQ ID NO.:28), and FIG. 1N)Peptide: LMEKEDYHSLY (SEQ ID NO.: 387).

FIGS. 2A through 2P show exemplary expression profile of source genes ofthe present invention that are over-expressed in different cancersamples. Tumor (black dots) and normal (grey dots) samples are groupedaccording to organ of origin. Box-and-whisker plots represent medianFPKM value, 25th and 75th percentile (box) plus whiskers that extend tothe lowest data point still within 1.5 interquartile range (IQR) of thelower quartile and the highest data point still within 1.5 IQR of theupper quartile. Normal organs are ordered according to risk categories.FPKM: fragments per kilobase per million mapped reads. Normal samples:blood cells; bloodvess (blood vessels); brain; heart; liver; lung;adipose (adipose tissue); adrenal gl (adrenal gland); bile duct;bladder; bone marrow, esoph (esophagus); eye; gall bl (gallbladder);head&neck; intest. la (large intestine); intest. sm (small intestine);kidney; lymph node; nerve periph (peripheral nerve); pancreas; parathyr(parathyroid gland); petit (peritoneum); pituit (pituitary); pleura;skel. mus (skeletal muscle); skin; spleen; stomach; thyroid; trachea;ureter; breast; ovary; placenta; prostate; testis; thymus; uterus. Tumorsamples: AML (acute myeloid leukemia); BRCA (breast cancer); CCC(cholangiocellular carcinoma); CLL (chronic lymphocytic leukemia); CRC(colorectal cancer); GBC (gallbladder cancer); GBM (glioblastoma); GC(gastric cancer); HCC (hepatocellular carcinoma); HNSCC (head and necksquamous cell carcinoma); MEL (melanoma); NHL (non-Hodgkin lymphoma);NSCLCadeno (non-small cell lung cancer adenocarcinoma); NSCLCother(NSCLC samples that could not unambiguously be assigned to NSCLCadeno orNSCLCsquam); NSCLCsquam (squamous cell non-small cell lung cancer); OC(ovarian cancer); OSCAR (esophageal cancer); other (other cancers, e.g.multiple myeloma), PACA (pancreatic cancer); PRCA (prostate cancer); RCC(renal cell carcinoma); SCLC (small cell lung cancer); UBC (urinarybladder carcinoma); UEC (uterine and endometrial cancer).

FIG. 2A) Gene symbol: MAGEA3, Peptide: VDPIGHLY (SEQ ID No.: 2), FIG.2B) Gene symbol: SLC6A3, Peptide: FGTTPAAEYF (SEQ ID No.:3), FIG. 2C)Gene symbol: UMODL1, Peptide: ARDPITFSF (SEQ ID No.: 6), FIG. 2D) Genesymbol: SLC45A3, Peptide: ASDHWRGRY (SEQ ID No.: 11), FIG. 2E) Genesymbol: MAGEA4, Peptide: VDPASNTY (SEQ ID No.: 86), FIG. 2F) Genesymbol: SSX1, Peptide: AFDDIATYF (SEQ ID No.: 87), FIG. 2G) Gene symbol:MAGEA1, Peptide: EVYDGREHSAY (SEQ ID No.: 89), FIG. 2H) Gene symbol:MMP12, Peptide: SSDPKAVMF (SEQ ID No.: 95), FIG. 2I) Gene symbol:UMODL1, Peptide: ASDDVRIEVGLY (SEQ ID No.: 7), FIG. 2J) Gene symbol:C7orf72, Peptide: TSRAANIPGY (SEQ ID No.: 8), FIG. 2K) Gene symbol:CTCFL, Peptide: NTHTGTRPY (SEQ ID No.: 13), FIG. 2L) Gene symbol: SOX14,Peptide: DTDPLKAAGL (SEQ ID No.: 23), FIG. 2M) Gene symbol: HAS2, HAS3,Peptide: IATVIQLFY (SEQ ID No.: 66), FIG. 2N) Gene symbol: SLC6A3,Peptide: CLVLVIVLLY (SEQ ID No.: 91), FIG. 2O) Gene symbol: UMODL1,Peptide: TATLLIVRY (SEQ ID No.: 96), and FIG. 2P) Gene symbol: CDK6,Peptide: LTSVVVTLW (SEQ ID No.: 374).

FIGS. 3A through 3G show exemplary results of peptide-specific in vitroCD8+ T cell responses of a healthy HLA-A*01+ donor. CD8+ T cells wereprimed using artificial APCs coated with anti-CD28 mAb and HLA-A*01 incomplex with SeqID No 417 peptide (KLDRSVFTAY) (FIG. 3A, left panel),Seq ID NO: 429 peptide (VSDSECLSRY) (FIG. 3B, left panel), Seq ID NO: 19peptide (LTEGHSGNY) (FIG. 3C, left panel), Seq ID NO: 33 peptide(SMDPVTGYQY) (FIG. 3D, left panel), Seq ID NO: 61 peptide (SSDIVALGGFLY)(FIG. 3E, left panel), Seq ID NO: 77 peptide (FTSDTGLEY) (FIG. 3F, leftpanel), or Seq ID NO: 83 peptide (DTEFHGGLHY) (FIG. 3G, left panel).After three cycles of stimulation, the detection of peptide-reactivecells was performed by 2D multimer staining with A*01/SeqID No 417 (FIG.3A), or A*01/SeqID No 429 (FIG. 3B), A*01/SeqID No 19 (FIG. 3C),A*01/SeqID No 33 (FIG. 3D), A*01/SeqID No 61 (FIG. 3E), A*01/SeqID No 77(FIG. 3F), or A*01/SeqID No 83 (FIG. 3G). Right panels show controlstaining of cells stimulated with irrelevant A*01/peptide complexes.Viable singlet cells were gated for CD8+ lymphocytes. Boolean gateshelped excluding false-positive events detected with multimers specificfor different peptides. Frequencies of specific multimer+ cells amongCD8+ lymphocytes are indicated.

EXAMPLES Example 1

Identification and Quantitation of Tumor Associated Peptides Presentedon the Cell Surface

Tissue Samples

Patients' tumor tissues were obtained from: Asterand (Detroit, Mich.,USA & Royston, Herts, UK), Bio-Options Inc. (Brea, Calif., USA),BioServe (Beltsville, Md., USA), Geneticist Inc. (Glendale, Calif.,USA), Leiden University Medical Center (LUMC) (Leiden, Netherlands),ProteoGenex Inc. (Culver City, Calif., USA); Saint Savas Hospital(Athens, Greece), Tissue Solutions Ltd (Glasgow, UK), UniversityHospital Bonn (Bonn, Germany), University Hospital Geneva (Geneva,Switzerland), University Hospital Heidelberg (Heidelberg, Germany),Osaka City University (OCU) (Osaka, Japan), University Hospital Tubingen(Tubingen, Germany). Normal tissues were obtained from Asterand(Detroit, Mich., USA & Royston, Herts, UK), BioServe (Beltsville, Md.,USA), Capital BioScience Inc. (Rockville, Md., USA), Centre for ClinicalTransfusion Medicine Tuebingen (Tubingen, Germany), Geneticist Inc.(Glendale, Calif., USA), ProteoGenex Inc. (Culver City, Calif., USA),Tissue Solutions Ltd (Glasgow, UK), University Hospital Heidelberg(Heidelberg, Germany), University Hospital Tubingen (Tubingen, Germany).

Written informed consents of all patients had been given before surgeryor autopsy. Tissues were shock-frozen immediately after excision andstored until isolation of TUMAPs at −70° C. or below.

Isolation of HLA Peptides from Tissue Samples

HLA peptide pools from shock-frozen tissue samples were obtained byimmune precipitation from solid tissues according to a slightly modifiedprotocol (Falk et al., 1991; Seeger et al., 1999) using theHLA-A*02-specific antibody BB7.2, the HLA-A, —B, C-specific antibodyW6/32, the HLA-DR specific antibody L243 and the HLA DP specificantibody B7/21, CNBr-activated sepharose, acid treatment, andultrafiltration.

Mass Spectrometry Analyses

The HLA peptide pools as obtained were separated according to theirhydrophobicity by reversed-phase chromatography (nanoAcquity UPLCsystem, Waters) and the eluting peptides were analyzed in LTQ-velos andfusion hybrid mass spectrometers (ThermoElectron) equipped with an ESIsource. Peptide pools were loaded directly onto the analyticalfused-silica micro-capillary column (75 μm i.d.×250 mm) packed with 1.7μm C18 reversed-phase material (Waters) applying a flow rate of 400 nLper minute. Subsequently, the peptides were separated using a two-step180 minute-binary gradient from 10% to 33% B at a flow rate of 300 nLper minute. The gradient was composed of Solvent A (0.1% formic acid inwater) and solvent B (0.1% formic acid in acetonitrile). A gold coatedglass capillary (PicoTip, New Objective) was used for introduction intothe nanoESl source. The LTQ-Orbitrap mass spectrometers were operated inthe data-dependent mode using a TOP5 strategy. In brief, a scan cyclewas initiated with a full scan of high mass accuracy in the orbitrap(R=30 000), which was followed by MS/MS scans also in the orbitrap(R=7500) on the 5 most abundant precursor ions with dynamic exclusion ofpreviously selected ions. Tandem mass spectra were interpreted bySEQUEST at a fixed false discovery rate (q0.05) and additional manualcontrol. In cases where the identified peptide sequence was uncertain itwas additionally validated by comparison of the generated naturalpeptide fragmentation pattern with the fragmentation pattern of asynthetic sequence-identical reference peptide.

Label-free relative LC-MS quantitation was performed by ion countingi.e. by extraction and analysis of LC-MS features (Mueller et al.,2007). The method assumes that the peptide's LC-MS signal areacorrelates with its abundance in the sample. Extracted features werefurther processed by charge state deconvolution and retention timealignment (Mueller et al., 2008; Sturm et al., 2008). Finally, all LC-MSfeatures were cross-referenced with the sequence identification resultsto combine quantitative data of different samples and tissues to peptidepresentation profiles. The quantitative data were normalized in atwo-tier fashion according to central tendency to account for variationwithin technical and biological replicates. Thus, each identifiedpeptide can be associated with quantitative data allowing relativequantification between samples and tissues. In addition, allquantitative data acquired for peptide candidates was inspected manuallyto assure data consistency and to verify the accuracy of the automatedanalysis. For each peptide a presentation profile was calculated showingthe mean sample presentation as well as replicate variations. Theprofiles juxtapose acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer samples to a baseline of normal tissuesamples.

Presentation profiles of exemplary over-presented peptides are shown inFIGS. 1A-1N.

Table 8 shows the presentation on various cancer entities for selectedpeptides, and thus the particular relevance of the peptides as mentionedfor the diagnosis and/or treatment of the cancers as indicated (e.g.peptide SEQ ID No. 21 for breast cancer and prostate cancer, peptide SEQID No. 47 for melanoma, non-small cell lung cancer, and ovarian cancer).

TABLE 8Overview of presentation of selected tumor-associated peptides of thepresent invention across entities. AML: acute myeloid leukemia; BRCA: breastcancer; CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia;CRC: colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC:gastric cancer; HCC: hepatocellular carcinoma; HNSCC: head and necksquamous cell carcinoma; MEL: melanoma; NHL: non-Hodgkin lymphoma;NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother:NSCLC samples that could not unambiguously be assigned to NSCLCadeno orNSCLCsquam; NSCLCsquam: squamous cell non-small cell lung cancer; OC:ovarian cancer; OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA:prostate cancer; RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC:urinary bladder carcinoma; UEC: uterine and endometrial cancer. SEQID No. Sequence Peptide Presentation on cancer entities 1 TLDSTRTLY PRCA2 VDPIGHLY HCC 3 FGTTPAAEYF OSCAR 4 RIEAIRAEY MEL 5 FMVIAGMPLFYNSCLCsquam 6 ARDPITFSF HNSCC, NSCLCadeno 7 ASDDVRIEVGLY AML, NSCLCother8 TSRAANIPGY MEL 9 QLDSTLDSY BRCA 10 VSERTGISY MEL 11 ASDHWRGRY PRCA 12YTDFVGEGLY BRCA, PRCA 13 NTHTGTRPY NSCLCsquam 14 QSEKEPGQQY HCC 15YLDSSKPAVY BRCA, UEC 16 NSDISIPEY BRCA, GC, NHL, OC, SCLC, UEC 17ASWAVLCYY NSCLCsquam 18 RSDPVSLRY GBM 19 LTEGHSGNY NHL 20 LSAQHRMLA GBM21 LSSAVNPIIY MEL 22 VMDTLGLFY BRCA, MEL, PRCA 23 DTDPLKAAGLGBC, GBM, GC, MEL, NSCLCadeno, NSCLCsquam, OC, OSCAR, UEC 24 NLDHYTNAYUEC 25 AMMQEAQLAY NSCLCadeno, NSCLCother, SCLC, UEC 26 ASDDFRSKY OSCAR27 PSEVPVDSHY GBM, MEL 28 PSEVPVDSHYY CCC, HCC, HNSCC, MEL, NSCLCadeno,NSCLCother, OC 29 TLEDLDNLYNY NSCLCadeno 30 VTTDKPRAY HNSCC 31VSDHLQAGMLG HNSCC, OSCAR QY 32 GTDKQNSTLRY MEL, NSCLCadeno 33 SMDPVTGYQYMEL 34 SSWSAGENDSY BRCA, CCC, GBC, GBM, GC, HNSCC, MEL,NSCLCsquam, OC, OSCAR, PACA, UEC 35 SWSAGENDSYSBRCA, NSCLCadeno, NSCLCsquam, UEC 36 MTSTEQSLY HCC, MEL 37 MTSTEQSLYYMEL 38 KSWSQSSSLMY HCC, HNSCC, MEL 39 WSQSSSLMYAML, GBC, GC, HCC, NSCLCsquam 40 TSDQLGYSY MEL 41 HSDLLEDSKYAML, BRCA, CLL, GBC, GC, HCC, HNSCC, MEL,NHL, NSCLCadeno, NSCLCother, NSCLCsquam,OC, OSCAR, PACA, PRCA, SCLC, UBC, UEC 42 ASDVDTLLK GBM 43 ETEPERHLGSYPRCA 44 IPSFNEMVY GBM, NSCLCadeno, NSCLCsquam 45 NLDPNKIY MEL, OSCAR 46RSDPGGGGLAY AML, BRCA AAY 47 WSDGVPLLY GBM, MEL 50 ITDEDEDMLSY HCC 53YLEDRPLSQLY MEL, OC 54 EVDIHTIHY GBC, HNSCC, OSCAR 55 ATEGDVLNYMEL, NHL, NSCLCsquam 56 VTEYAEEIYQY AML 57 ASDPASSTSCY HCC 58 YLENSASWYUEC 59 FTDSQGNDIK MEL 60 MTEKFLFLY HNSCC, OSCAR 61 SSDIVALGGFLYAML, BRCA, NSCLCsquam 62 VSELVTTGHY AML, CRC, GBC, GBM, NSCLCadeno,NSCLCsquam, OC, OSCAR 63 TSEISQNALMY NSCLCadeno, NSCLCother, NSCLCsquam64 TSEISQNALMYY NSCLCadeno 65 SSDFDPLVY BRCA, GBC, GBM, GC, HCC, MEL,NSCLCadeno, NSCLCsquam, UEC 67 NVDQNQNSY GBM, HNSCC, MEL, NSCLCadeno 68QSLPEFGLTY CCC, OSCAR 69 QSLPEFGLTYY OSCAR 70 YTELVEEKYBRCA, CCC, CRC, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, RCC, UEC 71LTDSTTRTTY CRC, GC, NSCLCadeno, NSCLCsquam, PACA 72 VTDSTTKIAYGC, NSCLCsquam, PACA, SCLC 73 STDSASYY HCC 74 EMEQQSQEY HNSCC, OSCAR 75FTDYELKAY HCC 76 QTDVERIKDTY CCC, CRC, GBC, GC, HCC, HNSCC, NHL,NSCLCadeno, NSCLCother, NSCLCsquam, OC, OSCAR, PACA, PRCA, UEC 77FTSDTGLEY AML, NSCLCadeno 78 QLDSAVKNLYBRCA, CRC, GBC, GC, HCC, HNSCC, MEL, NHL,NSCLCadeno, NSCLCsquam, OSCAR, PACA, SCLC, UBC, UEC 79 ASDLEPRELLSY BRCA80 ELCPLPGTSAY GBC, GBM, GC, HCC, HNSCC 81 YSDLHTPGRY AML, SCLC 82LTEKSHIRY BRCA, GBM, MEL, NSCLCadeno, NSCLCsquam 83 DTEFHGGLHYBRCA, GC, OSCAR, PACA 84 ESEMIKFASYY HCC 85 SSDNYEHWLYBRCA, MEL, NSCLCadeno 87 AFDDIATYF SCLC 88 KEVDPAGHSYI NHL 89EVYDGREHSAY MEL, OSCAR 90 YEDHFPLLF MEL, OSCAR 91 CLVLVIVLLY NSCLCadeno92 TTDDTTAMASAS GBC, MEL, NSCLCsquam 93 HLKILSPIY BRCA 95 SSDPKAVMF CRC96 TATLLIVRY AML 97 FPAPPAHWFY BRCA 98 NFSDLVFTY BRCA 99 AADSNPSEL PRCA100 TTSSAISWILY BRCA 101 SITDVDFIY UEC 102 STIRGELFF BRCA 103 ITDTLIHLMBRCA 104 ITDTLIHL BRCA 107 TTENSGNYY CLL 108 NSNLKFLEV SCLC 109ISEDKSISF CRC 111 TPIPFDKILY OSCAR 112 KASSVSAEDGY GBM, UEC 113ASCRSSAEY OC 114 AVAAAAGASLY AML, MEL, OSCAR 115 NEIDIHSIYFY OSCAR 116RSDIGEFEW AML 117 SPAKQFNIY GBC 119 TVFDENLSRY OSCAR 120 LVDENQSWY NHL121 SADEAHGLL MEL 122 ISEAPLTEV MEL 123 LLKAKDILY HNSCC 124 FLKVTGYDKDDYHCC 125 FQYELRELY OC 126 TTDPKKFQY MEL, NSCLCsquam, OC 127 VPFNLITEY MEL128 YTEFVDATFTK HNSCC 130 YIGLKGLYF MEL 131 LEDGIEQSAY NHL 132 RTHIGYKVYNSCLCsquam 134 SAPSSSGSPLY NSCLCsquam 135 TFDKQIVLL NSCLCother 136RRLNFSGFGY SCLC 137 EAYLERIGY BRCA 138 IPVHDSVGVTY MEL 139 PVHDSVGVTYGC, SCLC, UEC 140 SQHIFTVSY GBM, NSCLCsquam 141 DAVAPGREY GBC 142IEKFAVLY HNSCC 143 HVSGQMLYF BRCA 144 RTIEGDFLW GBM 145 LSDAVHVEF CLL146 LCATVCGTEQY HNSCC 147 AQVQDTGRY MEL 148 GTKQWVHARY CLL 149PIMSSSQALY NSCLCsquam 150 FTTLSDLQTNMA HNSCC, MEL, NHL 151 YEVDTKLLSLMEL 155 HTMEVTVY MEL 156 STALSILLL MEL 157 GLIEVVTGY MEL, OC 158EVTDRNMLAF HNSCC, OC, OSCAR 159 RQAPGPARDY NSCLCsquam 160 EVLGEEMYAYPRCA 161 EAAPDIMHY MEL, OSCAR 162 IADNPQLSFY BRCA 163 KIRAEVLSHY CLL 164KLAGTVFQY NSCLCsquam 165 VSVYNSYPY BRCA 166 YHRICELLSDY GC 167 RAVQPGETYNSCLCother, NSCLCsquam 168 VQPGETYTY GBC 169 TVDNANILL UBC 170 VQIAKGMNYNSCLCsquam 171 ITDFGLAKL NSCLCadeno 172 FSEPFHLIV MEL 173 QSTTGVSHY CCC174 TSEVEGLAFVSY NSCLCsquam 175 GLEYEAPKLY GBM 176 HTDLESPSAVY HCC, UBC178 TQRTSFQFY NSCLCadeno, NSCLCsquam, OC 179 SSTDFTFASW HNSCC, OC, OSCAR180 AQISDTGRY MEL 181 SVTDLIGGKW OSCAR 182 TQPELSSRY NSCLCadeno 183LADTDLGMTF OSCAR 184 KTIQEVAGY GBM, HNSCC 185 NSDESADSEPH BRCA KY 186AVSSGLFFY BRCA 187 TQKSVQVLAY GBM 189 FRGVFVHRY MEL 190 VSSTVHNLY BRCA191 FTRAFDQLRM NSCLCadeno 192 LAFYYGMYGC, MEL, NHL, NSCLCsquam, OC, PRCA, RCC, UEC 193 SQNGQLIHY OC 194CYTADNEMGY HCC 195 YTADNEMGYY NSCLCadeno, NSCLCother 196 RLAQYTIERY HCC197 NDEIDKLTGY MEL 198 KLTDYINANY GBM 199 LCAAVLAKY MEL 200 SLPEFGLTYGBM, RCC 201 SLPEFGLTYY OSCAR 202 QTDINGGSLK MEL 203 LSQDELSKFNSCLCsquam 204 NVKEAPTEY NSCLCother 205 RMQEGSEVY MEL 206 RVFVAVTLYOC, PRCA 207 LLEGEDAHLTQY HNSCC 208 LLISKAEDY MEL 209 EADPFLKYL AML 210LLEADPFLKY AML 211 YLNEWGSRF NSCLCadeno 212 MMTDLTSVY MEL 213 VSDSTTEITYPACA 214 VQDPSLPVY NSCLCsquam 215 DTLEAATSLY HCC 216 NSMLDPLVYBRCA, HNSCC, NSCLCsquam, UBC 217 LMDEGAVLTL HNSCC 218 FTAQLQLY GBC 219KTELETALYY HCC 220 DVERIKDTY NSCLCsquam 221 TDVERIKDTY HCC, NSCLCsquam222 GSPDAVVSY NSCLCsquam 223 NAVDVVPSSF GBC 224 RTDEGDNRVW OSCAR 226QITPKHNGLY NSCLCsquam, OC 228 KSFDDIAKY OC 229 MTDVFIDY NSCLCsquam 230CVIETFHKY GBC 231 LLPLLVMAY OC 232 RYLNIVHATQLY CLL 233 RINSATGQY CLL234 YTDLTTIQV HNSCC 235 SIEIDHTQY CCC, MEL, NSCLCadeno 236 VLDSLLAQY GC237 AQEAAVFLTLY SCLC 238 ETDWGLLKGHT BRCA Y 239 SSERGSPIEKY OSCAR 240EVLDSLLAQY OSCAR 241 SLMVASLTY OC 242 GTNLPTLLW HCC, OSCAR 243 LTSEDTGAYNSCLCadeno 244 VTKYIAGPY BRCA 245 LSDNAANRY AML 246 ARLEGEIATYNSCLCsquam 247 SMIRVGTNY NSCLCsquam, OC 248 VTDIDELGK GBM 249 GVGFTELEYGC 250 GYVCNACGLY HNSCC 251 GIEMTYETY GBC 252 DTTSHTYLQY OSCAR 253YLESHGLAY NSCLCsquam 254 FLFNDALLY BRCA 255 WELDSLEY GBM 256 HAFESNNFIYGBC 257 KSEMNVNMKY BRCA, GBC, NSCLCsquam, PRCA, UEC 258 RPSSVLTIYGBM, MEL, NSCLCsquam 259 APDEVVALL MEL 260 KPTEDSANVY OSCAR 261MTEGSTVNTEY GBC, GC 263 DCMDTEGSYM UEC 264 YRDPVFVSL OC 266 LTDSFLLRFHCC 267 IVADDTVY NSCLCadeno, NSCLCother 268 AILHHLYFY BRCA 269LPSPAATIWDY GBC 270 DLKIDLAAQY OSCAR 271 VAEPPVVCSY HCC 272 IPQDECLRYGBC, HNSCC 273 CGPNEINHFY NSCLCsquam 274 YADIHGDLL RCC 275 ESDEMENLLTYBRCA 276 QITSFASGTSY GC 277 LPAPGFLFY GBC 278 AATVKSDIY NSCLCsquam 279LMTVLLKY BRCA 280 TTEMVSNESVDY GBC 281 YPDLSELLM AML, RCC, UEC 282QAMPSWPTAAY NSCLCsquam 283 ETILVSSSY NSCLCadeno 284 TCSHTFVYYNSCLCadeno, SCLC 285 VLPHHSEGACVY NSCLCadeno, OC 286 ATDMEGNLNYBRCA, GBM, NSCLCadeno 287 ENSIEDLQY HCC 289 YTSHEDIGY NSCLCadeno 290GQFTGTAGACR NSCLCadeno, OC Y 291 TSDVTGSLTYBRCA, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother 292 VLDFAPPGASAY OC 293IISVLIAIY BRCA 294 MMEMEGMY NSCLCsquam 295 GQRLDEAMISY OC 296 HMLAAMAYBRCA, MEL, NHL, NSCLCadeno, NSCLCother, NSCLCsquam, OC, RCC 297RLDEAMISY UBC 298 KFDVINHYF BRCA 299 EVDSVALSL MEL 300 VSINPNSGDIY GC301 ESQTCASDY GBC, NSCLCother 302 FYLSTPENYHY BRCA 303 GFGGLSSQGVYCCC, HNSCC Y 304 FSENLIYTYI MEL 305 YADLLIYTY NSCLCadeno, UBC 306KSFETTVRY MEL, OSCAR 307 DTDDRELRY GBM, HCC, UEC 308 ELAAGQVVYCLL, OSCAR 309 EVDRNLIQY UEC 311 TVTDGTHTDFY NSCLCsquam 312 VTDGINPLIOSCAR 313 VTDGTHTDFY MEL 314 PPEANSLQGALY CCC, OC 315 VLKIELETYGBM, GC, NSCLCadeno, NSCLCsquam, OC, PACA, PRCA, RCC 316 YTCEECGQAFBRCA, GBM, GC, HCC, HNSCC, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, UEC317 EDLLEVLDMY UEC 318 YMTSMALNY NSCLCsquam 319 FTDPHIITF BRCA 320QALQDKLQTFY BRCA 321 DGIADASNLSYY MEL, NSCLCadeno, NSCLCsquam 322FSELNPLALY GC, HNSCC, NSCLCsquam, OSCAR 323 KTLQKPVLPLY CLL 324RTGIFPYRF GBC 325 LQKPVLPLY CLL, NSCLCsquam 326 STSRLTLFS GBM, HNSCC 327IMLSVDQHLY HNSCC, MEL 328 LLDEDNNIKL GBC 329 NTDSMTLNNTAYBRCA, GBC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR, UBC 331 YLYQAPGSLALYNSCLCadeno 332 SLISFKYTSY OC 333 LSDPQAELQFY GBM 334 PSSMPECLSYNSCLCsquam 335 PSSMPECLSYY NSCLCadeno 336 ATNIQLNIDTY OC 337 FTESNQYNIYBRCA, MEL, NSCLCadeno, NSCLCsquam 338 YSPDSFNVSW OSCAR 339 ESMDIFPLGWHNSCC, OC, OSCAR 340 SVDSNLVAY NSCLCadeno 341 PANYLGKMTYMEL, NSCLCadeno, OC, PRCA 343 YFGNYFTYY BRCA 344 AVNALQSVYAML, GBC, GBM, GC, NSCLCadeno, NSCLCother, NSCLCsquam, OC, PACA, PRCA,UEC 345 NTMDAVPRIDHY GBC, GC 346 VAGLEAGVLYBRCA, GBM, HCC, MEL, OC, UBC, UEC 347 SADHPGLTF CRC 349 HLLSVSLYYNSCLCsquam 350 LTDPQVSYV MEL 351 VLDPMLDFY HCC 352 YPVVVAESMY MEL 353RLNGSVASY OC 355 MADRGEARL AML 356 NSENHILKY GC, OSCAR 358 YMSPDIALLYGBM, HCC, NSCLCadeno 359 NKEINYFMY CCC 360 RFDDINQEF HNSCC 361 FTAEEGQLYBRCA, GBC, GBM, HCC, NSCLCadeno,NSCLCother, NSCLCsquam, PACA, PRCA, RCC, SCLC, UBC, UEC 362 SGALDEAAAYGBM, NSCLCadeno, PRCA 363 LTDRDVSFY HCC 364 DTGYLQLYY BRCA 365 FVDTKVPEHHCC, NHL, NSCLCsquam 366 ITVDVRDEF GBM 367 LTDTGYLQLYGBM, HNSCC, OC, OSCAR, UBC 368 ESAATGQLDY OC 369 AVMEAAFVY BRCA 370RLSTIRHLY HNSCC, MEL 371 WSDSTSQTIY OSCAR 372 SRSDFEWVY BRCA, OSCAR 373FHADSDDESF NHL 374 LTSVVVTLW HCC 375 ASSLDSLHY BRCA 376 EDDEDEDLYNSCLCsquam 377 YADPSANRDLL OC 378 TAKAPSTEYGBC, NSCLCadeno, NSCLCother, NSCLCsquam 379 SLIIDDTEY GBC, NSCLCsquam380 VACGNNPVY NSCLCadeno 381 ETSFSTSHY HNSCC 382 YEPATMEQY NSCLCsquam383 PPDHAVGRTKY UBC 384 RFRSITQSYY CLL 385 SANALILTY BRCA, MEL 386NSALNPLLY BRCA, NSCLCadeno 387 LMEKEDYHSLY MEL 388 YTAHVGYSMY NSCLCadeno389 YYDLVESTF AML 390 FSEPFHLIVSY CLL, MEL 392 TQHFVQENY MEL 393QVWGGQPVY MEL 394 QVPLDCVLY HNSCC, OSCAR 395 ILKGGSGTY MEL 396 LPDPNVQKYMEL 397 NSAINPLIY NSCLCadeno, RCC, UEC 398 YYYDTHTNTY HNSCC, PRCA

Example 2

Expression Profiling of Genes Encoding the Peptides of the Invention

Over-presentation or specific presentation of a peptide on tumor cellscompared to normal cells is sufficient for its usefulness inimmunotherapy, and some peptides are tumor-specific despite their sourceprotein occurring also in normal tissues. Still, mRNA expressionprofiling adds an additional level of safety in selection of peptidetargets for immunotherapies. Especially for therapeutic options withhigh safety risks, such as affinity-matured TCRs, the ideal targetpeptide will be derived from a protein that is unique to the tumor andnot found on normal tissues.

RNA Sources and Preparation

Surgically removed tissue specimens were provided as indicated above(see Example 1) after written informed consent had been obtained fromeach patient. Tumor tissue specimens were snap-frozen immediately aftersurgery and later homogenized with mortar and pestle under liquidnitrogen. Total RNA was prepared from these samples using TRI Reagent(Ambion, Darmstadt, Germany) followed by a cleanup with RNeasy (QIAGEN,Hilden, Germany); both methods were performed according to themanufacturer's protocol.

Total RNA from healthy human tissues for RNASeq experiments was obtainedfrom: Asterand (Detroit, Mich., USA & Royston, Herts, UK); Bio-OptionsInc. (Brea, Calif., USA); Geneticist Inc. (Glendale, Calif., USA);ProteoGenex Inc. (Culver City, Calif., USA); Tissue Solutions Ltd(Glasgow, UK).

Total RNA from tumor tissues for RNASeq experiments was obtained from:Asterand (Detroit, Mich., USA & Royston, Herts, UK); BioCat GmbH(Heidelberg, Germany); BioServe (Beltsville, Md., USA); Geneticist Inc.(Glendale, Calif., USA); Istituto Nazionale Tumori “Pascale” (Naples,Italy); ProteoGenex Inc. (Culver City, Calif., USA); University HospitalHeidelberg (Heidelberg, Germany).

Quality and quantity of all RNA samples were assessed on an Agilent 2100Bioanalyzer (Agilent, Waldbronn, Germany) using the RNA 6000 PicoLabChip Kit (Agilent).

RNAseq Experiments

Gene expression analysis of—tumor and normal tissue RNA samples wasperformed by next generation sequencing (RNAseq) by CeGaT (Tubingen,Germany). Briefly, sequencing libraries are prepared using the IlluminaHiSeq v4 reagent kit according to the provider's protocol (IlluminaInc., San Diego, Calif., USA), which includes RNA fragmentation, cDNAconversion and addition of sequencing adaptors. Libraries derived frommultiple samples are mixed equimolar and sequenced on the Illumina HiSeq2500 sequencer according to the manufacturer's instructions, generating50 bp single end reads. Processed reads are mapped to the human genome(GRCh38) using the STAR software. Expression data are provided ontranscript level as RPKM (Reads Per Kilobase per Million mapped reads,generated by the software Cufflinks) and on exon level (total reads,generated by the software Bedtools), based on annotations of the ensemblsequence database (Ensembl77). Exon reads are normalized for exon lengthand alignment size to obtain RPKM values.

Exemplary expression profiles of source genes of the present inventionthat are highly over-expressed or exclusively expressed in acute myeloidleukemia, breast cancer, cholangiocellular carcinoma, chroniclymphocytic leukemia, colorectal cancer, gallbladder cancer,glioblastoma, gastric cancer, hepatocellular carcinoma, head and necksquamous cell carcinoma, melanoma, non-Hodgkin lymphoma, lung cancer(including non-small cell lung cancer adenocarcinoma, squamous cellnon-small cell lung cancer, and small cell lung cancer), ovarian cancer,esophageal cancer, pancreatic cancer, prostate cancer, renal cellcarcinoma, urinary bladder carcinoma, uterine and endometrial cancer areshown in FIGS. 2A-2P. Expression scores for further exemplary genes areshown in Table 9.

TABLE 9Expression scores. The table lists peptides from genes that are veryhighly over-expressed in tumors compared to a panel of normal tissues (+++),highly over-expressed in tumors compared to a panel of normal tissues (++) orover-expressed in tumors compared to a panel of normal tissues (+). The baselinefor this score was calculated from measurements of the following relevant normaltissues: adipose tissue, adrenal gland, bile duct, blood cells, blood vessels, bonemarrow, brain, esophagus, eye, gallbladder, heart, head and neck, kidney, largeintestine, liver, lung, lymph node, nerve, parathyroid, pancreas, pituitary, pleura,skeletal muscle, skin, small intestine, spleen, stomach, thyroid gland, trachea,ureter, urinary bladder. In case expression data for several samples of the sametissue type were available, the arithmetic mean of all respective samples wasused for the calculation. AML: acute myeloid leukemia; BRCA: breast cancer;CCC: cholangiocellular carcinoma; CLL: chronic lymphocytic leukemia; CRC:colorectal cancer; GBC: gallbladder cancer; GBM: glioblastoma; GC: gastriccancer; HCC: hepatocellular carcinoma; HNSCC: head and neck squamous cellcarcinoma; MEL: melanoma; NHL: non-Hodgkin lymphoma; NSCLCadeno: non-small cell lung cancer adenocarcinoma; NSCLCother: NSCLC samples that couldnot unambiguously be assigned to NSCLCadeno or NSCLCsquam;NSCLCsquam: squamous cell non-small cell lung cancer; OC: ovarian cancer;OSCAR: esophageal cancer; PACA: pancreatic cancer; PRCA: prostate cancer;RCC: renal cell carcinoma; SCLC: small cell lung cancer; UBC: urinary bladdercarcinoma; UEC: uterine and endometrial cancer.Gene Expression in tumor samples highly over- very highly over-Seq ID No Sequence over-expressed (+) expressed (++) expressed (+++)   1TLDSTRTLY GBM PRCA   2 VDPIGHLY CRC, NHL, OC, GBC, GC, HCC, PACANSCLCadeno, HNSCC, MEL, UEC NSCLCsquam, OSCAR, SCLC, UBC   3 FGTTPAAEYFNSCLCadeno, RCC NSCLCsquam, SCLC   4 RIEAIRAEY MEL   5 FMVIAGMPLFYNSCLCadeno, RCC NSCLCsquam   6 ARDPITFSF BRCA, CRC, OC, NSCLCadeno, AMLSCLC NSCLCother   7 ASDDVRIEVGLY BRCA, CRC, OC, NSCLCadeno, AML SCLCNSCLCother   8 TSRAANIPGY BRCA, CLL NHL   9 QLDSTLDSY OC BRCA  10VSERTGISY MEL  11 ASDHWRGRY PRCA  12 YTDFVGEGLY PRCA  13 NTHTGTRPYGBC, MEL, NHL OC  14 QSEKEPGQQY GBC HCC  15 YLDSSKPAVY BRCA, OC, UEC  16NSDISIPEY OC, UEC  17 ASWAVLCYY PRCA  18 RSDPVSLRY GBM  19 LTEGHSGNYCLL, NHL  20 LSAQHRMLA CLL, SCLC NHL  21 LSSAVNPIIY NSCLCsquam, GC, PACAUEC  22 VMDTLGLFY HCC PRCA  23 DTDPLKAAGL GBC, HCC, CRC, GC, PRCA HNSCC,NSCLCadeno, NSCLCother, SCLC, UBC, UEC  24 NLDHYTNAY SCLC, UEC  25AMMQEAQLAY GBM SCLC  26 ASDDFRSKY NSCLCsquam, HNSCC, MEL OSCAR, UBC  27PSEVPVDSHY CCC MEL  28 PSEVPVDSHYY CCC MEL  29 TLEDLDNLYNYCRC, GBC, NHL, BRCA, GC, UBC, UEC HNSCC, MEL, NSCLCadeno, NSCLCsquam,OC, OSCAR, PACA  30 VTTDKPRAY MEL  31 VSDHLQAGMLGQY NSCLCother, HNSCCOSCAR  32 GTDKQNSTLRY MEL  33 SMDPVTGYQY MEL  34 SSWSAGENDSY NHLHNSCC, MEL  35 SWSAGENDSYS NHL HNSCC, MEL  36 MTSTEQSLY BRCA, HCC, PRCAMEL, OC, SCLC, UEC  37 MTSTEQSLYY BRCA, HCC, PRCA MEL, OC, SCLC, UEC  38KSWSQSSSLMY CCC HCC  39 WSQSSSLMY CCC HCC  40 TSDQLGYSY MEL  41HSDLLEDSKY BRCA  42 ASDVDTLLK GBM  43 ETEPERHLGSY PRCA  44 IPSFNEMVY GBM 45 NLDPNKIY CRC, GC, MEL, GBC NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA 46 RSDPGGGGLAYAAY GBM, SCLC AML  47 WSDGVPLLY GBM  48 FTTQDELLVY PRCA 49 GSFSIQHTY PRCA  50 ITDEDEDMLSY GBC, HCC  51 STEERRLNY PRCA  52TTQDELLVY PRCA  53 YLEDRPLSQLY MEL  54 EVDIHTIHY OSCAR HNSCC  55ATEGDVLNY NHL CLL  56 VTEYAEEIYQY OC, OSCAR AML, UEC  57 ASDPASSTSCYNSCLCother  58 YLENSASWY UEC  59 FTDSQGNDIK MEL  60 MTEKFLFLYBRCA, CLL, NHL, UBC RCC  61 SSDIVALGGFLY BRCA, HNSCC NSCLCsquam,OSCAR, UBC  62 VSELVTTGHY GBM  63 TSEISQNALMY NSCLCother, NSCLCadenoNSCLCsquam  64 TSEISQNALMYY NSCLCother, NSCLCadeno NSCLCsquam  65SSDFDPLVY HCC CRC  66 IATVIQLFY HNSCC, UBC NSCLCsquam, OSCAR  67NVDQNQNSY MEL  68 QSLPEFGLTY GC, HNSCC, CRC OSCAR, UBC  69 QSLPEFGLTYYGC, HNSCC, CRC OSCAR, UBC  70 YTELVEEKY UEC  71 LTDSTTRTTY PACA  72VTDSTTKIAY PACA  73 STDSASYY HCC  74 EMEQQSQEY OSCAR HNSCC  75 FTDYELKAYHCC  76 QTDVERIKDTY CCC, GC, OSCAR HNSCC, NSCLCother, NSCLCsquam, UBC 77 FTSDTGLEY AML  78 QLDSAVKNLY CRC, NHL  79 ASDLEPRELLSY GBM  80ELCPLPGTSAY GBM, OC, SCLC  81 YSDLHTPGRY MEL, NSCLCadeno, SCLC  82LTEKSHIRY MEL  83 DTEFHGGLHY GBC, GC, PACA  84 ESEMIKFASYY HCC  85SSDNYEHWLY CLL  86 VDPASNTY BRCA, CRC, GC, HCC, GBC, HNSCC, UECNSCLCadeno MEL, NSCLCsquam, OC, OSCAR, SCLC, UBC  87 AFDDIATYF MEL, NHL,SCLC HCC NSCLCadeno  88 KEVDPAGHSYI CRC, PACA BRCA, GC, HCC, GBC, HNSCC,NHL, MEL, NSCLCadeno, NSCLCsquam, OC, UEC OSCAR, SCLC, UBC  89EVYDGREHSAY BRCA, CCC, OC, GBC, GC, HCC, MEL, UBC HNSCC, NHL,NSCLCsquam, NSCLCadeno, OSCAR SCLC  90 YEDHFPLLF HCC, GBC, GC, MEL, UBCNSCLCadeno, HNSCC, UEC NSCLCsquam, OC, OSCAR, SCLC  91 CLVLVIVLLYNSCLCadeno, RCC NSCLCsquam  92 TTDDTTAMASAS HCC, UEC GBC, GC, MEL, UBCHNSCC, NSCLCadeno, NSCLCsquam, OC, OSCAR, SCLC  93 HLKILSPIYBRCA, CRC, NHL, NSCLCadeno, AML OC, SCLC, UEC NSCLCother  94 KPSAVKDSIYOC PRCA, SCLC UEC  95 SSDPKAVMF HCC, RCC CCC, GC, MEL, CRC, GBC, NHL,HNSCC, NSCLCadeno, NSCLCother, OC, PACA, UEC NSCLCsquam, OSCAR, SCLC,UBC  96 TATLLIVRY BRCA, CRC, NSCLCadeno, AML SCLC NSCLCother  97FPAPPAHWFY OC BRCA  98 NFSDLVFTY CLL, HNSCC, BRCA, SCLC GBM MEL, NHL,NSCLCadeno, NSCLCsquam, OC, PACA, RCC, UBC  99 AADSNPSEL PRCA 100TTSSAISWILY BRCA 101 SITDVDFIY CRC, SCLC PRCA 102 STIRGELFFHCC, OC, RCC, BRCA, CCC, SCLC CRC, GBC, GC, HNSCC, NSCLCadeno,NSCLCsquam, OSCAR, PACA, UBC, UEC 103 ITDTLIHLM BRCA, OC, UEC 104ITDTLIHL BRCA, OC, UEC 105 VVFDKSDLAKY PRCA 106 EVVEGKEWGSFYCRC, GBC, GC, PACA 107 TTENSGNYY CLL, NHL 108 NSNLKFLEV BRCA, CRC, GC,HNSCC, MEL, NHL, NSCLCsquam, NSCLCadeno, OC NSCLCother, OSCAR, PACA,SCLC, UEC 109 ISEDKSISF MEL 110 IGDKVDAVY NSCLCadeno, HNSCC, UBCNSCLCsquam, OSCAR 111 TPIPFDKILY CRC, BRCA, CCC, NSCLCsquam, GBC, GC,OC, UBC, UEC HNSCC, NSCLCadeno, OSCAR, PACA 112 KASSVSAEDGY BRCA, SCLC,PRCA UBC 113 ASCRSSAEY GBC, GC, CCC, HNSCC, NSCLCadeno, NSCLCsquam,NSCLCother, OSCAR, UBC PACA, UEC 114 AVAAAAGASLY UEC 115 NEIDIHSIYFYNSCLCother, HNSCC OSCAR 116 RSDIGEFEW AML, BRCA, CLL, SCLCCCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, UBC, UEC 117 SPAKQFNIY AML, BRCA, CLL, SCLCCCC, CRC, GBC, GBM, GC, HCC, HNSCC, MEL, NHL, NSCLCadeno, NSCLCother,NSCLCsquam, OC, OSCAR, PACA, UBC, UEC 118 LTWAHSAKY CCC MEL 119TVFDENLSRY NSCLCother, HNSCC OSCAR 120 LVDENQSWY NSCLCother, HNSCC OSCAR121 SADEAHGLL BRCA, GC, CCC, CRC, GBC HNSCC, MEL, NHL, NSCLCadeno,NSCLCother, NSCLCsquam, OC, OSCAR, PACA, RCC, UBC, UEC 122 ISEAPLTEV MEL123 LLKAKDILY HCC, PRCA 124 FLKVTGYDKDDY MEL 125 FQYELRELY MEL 126TTDPKKFQY MEL 127 VPFNLITEY HCC MEL 128 YTEFVDATFTK OSCAR HNSCC 129STIDFRAGF MEL 130 YIGLKGLYF HCC MEL 131 LEDGIEQSAY AML CLL 132 RTHIGYKVYMEL 133 ITDVGPGNY MEL 134 SAPSSSGSPLY CLL SCLC 135 TFDKQIVLL CCC HCC 136RRLNFSGFGY SCLC 137 EAYLERIGY BRCA 138 IPVHDSVGVTY GBM 139 PVHDSVGVTYGBM 140 SQHIFTVSY GBM 141 DAVAPGREY CCC HCC 142 IEKFAVLY GBM 143HVSGQMLYF BRCA UEC 144 RTIEGDFLW GBM 145 LSDAVHVEF CLL, NHL 146LCATVCGTEQY BRCA GBM, MEL 147 AQVQDTGRY MEL 148 GTKQWVHARY CLL, NHL 149PIMSSSQALY MEL 150 FTTLSDLQTNMA AML, GBC, HCC, MEL, SCLC HNSCC,NSCLCadeno, OC, OSCAR 151 YEVDTKLLSL GBC, HCC, MEL, SCLC HNSCC,NSCLCadeno, NSCLCsquam, OC, OSCAR 152 YLEDRPLSQ MEL 153 HSIEVFTHY MEL154 SIEVFTHY MEL 155 HTMEVTVY MEL 156 STALSILLL GBM 157 GLIEVVTGY BRCA158 EVTDRNMLAF OC BRCA 159 RQAPGPARDY NSCLCsquam, HNSCC, OSCAR UBC 160EVLGEEMYAY BRCA PRCA 161 EAAPDIMHY BRCA, HCC, PRCA MEL, OC 162IADNPQLSFY PRCA BRCA 163 KIRAEVLSHY CLL 164 KLAGTVFQY BRCA, CRC, GC,CCC, GBC HNSCC, MEL, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA,SCLC, UBC 165 VSVYNSYPY PRCA 166 YHRICELLSDY BRCA, GBC, PRCA NSCLCsquam,OC, OSCAR, PACA, SCLC 167 RAVQPGETY CCC HCC 168 VQPGETYTY CCC HCC 169TVDNANILL NSCLCsquam, HNSCC, OSCAR UBC 170 VQIAKGMNY HNSCC GBM 171ITDFGLAKL HNSCC GBM 172 FSEPFHLIV MEL CLL, NHL 173 QSTTGVSHY GBM 174TSEVEGLAFVSY GBM 175 GLEYEAPKLY AML 176 HTDLESPSAVY UBC 177 LVDGKWQEFUBC 178 TQRTSFQFY NSCLCother, NSCLCadeno NSCLCsquam 179 SSTDFTFASWGC, HNSCC, CRC NSCLCsquam, OSCAR, UBC 180 AQISDTGRY MEL 181 SVTDLIGGKWAML, HNSCC, UBC NSCLCsquam, OSCAR 182 TQPELSSRY BRCA, CRC, GC, CCC, GBCHNSCC, NSCLCadeno, NSCLCother, NSCLCsquam, OSCAR, PACA, UBC 183LADTDLGMTF HNSCC, MEL, GBC NHL, NSCLCadeno, NSCLCother, SCLC 184KTIQEVAGY GBM 185 NSDESADSEPHKY NHL 186 AVSSGLFFY NHL CLL 187 TQKSVQVLAYGBM 188 DIPDYLLQY NHL 189 FRGVFVHRY MEL, GBC, NHL NSCLCadeno, OC, SCLC190 VSSTVHNLY BRCA, OC PRCA 191 FTRAFDQLRM CLL 192 LAFYYGMY GBM, HNSCC,NSCLCsquam NSCLCadeno, OSCAR 193 SQNGQLIHY MEL 194 CYTADNEMGYNSCLCother, NSCLCadeno NSCLCsquam 195 YTADNEMGYY NSCLCother, NSCLCadenoNSCLCsquam 196 RLAQYTIERY GBC, GC, UEC PACA 197 NDEIDKLTGY PRCA 198KLTDYINANY GBM 199 LCAAVLAKY HCC 200 SLPEFGLTY GC, HNSCC, CRC OSCAR, UBC201 SLPEFGLTYY GC, HNSCC, CRC OSCAR, UBC 202 QTDINGGSLK MEL 203LSQDELSKF BRCA, CCC, NHL, GBC NSCLCsquam, OC, OSCAR, UBC, UEC 204NVKEAPTEY NHL CLL 205 RMQEGSEVY NHL CLL 206 RVFVAVTLY PRCA 207LLEGEDAHLTQY NSCLCsquam, HNSCC, OSCAR UBC 208 LLISKAEDY BRCA 209EADPFLKYL OC, OSCAR, UEC AML 210 LLEADPFLKY OC, OSCAR, UEC AML 211YLNEWGSRF GBC, HNSCC NSCLCadeno, NSCLCsquam, OSCAR, UBC 212 MMTDLTSVYPACA 213 VSDSTTEITY PACA 214 VQDPSLPVY CLL, CRC, GBM, NHL GC, HNSCC,MEL, NSCLCadeno, NSCLCsquam, OC, OSCAR, PACA, PRCA, SCLC, UBC 215DTLEAATSLY NSCLCadeno, SCLC NSCLCsquam, OC 216 NSMLDPLVY UBC BRCA 217LMDEGAVLTL CLL, NHL CRC 218 FTAQLQLY NSCLCother, NSCLCadeno NSCLCsquam219 KTELETALYY GBM, HNSCC, CRC NSCLCsquam, OSCAR, PACA, RCC 220DVERIKDTY CCC, GC, OSCAR HNSCC, NSCLCother, NSCLCsquam, UBC 221TDVERIKDTY CCC, GC, OSCAR HNSCC, NSCLCother, NSCLCsquam, UBC 222GSPDAVVSY CCC, CRC, GBC, GC, HCC, HNSCC, MEL, NSCLCsquam, OC, OSCAR,PACA, SCLC 223 NAVDVVPSSF BRCA, CCC, GBC, NSCLCsquam, OC, OSCAR,UBC, UEC 224 RTDEGDNRVW GBC, HNSCC, NSCLCadeno, NSCLCother, NSCLCsquam,OC, OSCAR, UBC 225 STDPNIVRK MEL, PRCA, SCLC 226 QITPKHNGLY CCC, MEL 227ESAPKEVSRY RCC, UBC 228 KSFDDIAKY GBC, HCC, NHL, NSCLCadeno, OC, PACA229 MTDVFIDY HCC 230 CVIETFHKY HNSCC, MEL 231 LLPLLVMAY CLL 232RYLNIVHATQLY CLL 233 RINSATGQY CLL 234 YTDLTTIQV BRCA, HNSCC, MEL, NHL,NSCLCadeno, OSCAR 235 SIEIDHTQY HCC 236 VLDSLLAQY GBC, HCC, HNSCC,NSCLCsquam, SCLC 237 AQEAAVFLTLY PRCA 238 ETDWGLLKGHTY UEC 239SSERGSPIEKY BRCA 240 EVLDSLLAQY GBC, HCC, HNSCC, MEL, NSCLCsquam,OC, SCLC 241 SLMVASLTY AML, CCC, PACA, UEC 242 GTNLPTLLW SCLC 243LTSEDTGAY SCLC 244 VTKYIAGPY CCC 245 LSDNAANRY AML 246 ARLEGEIATYHNSCC, OSCAR 247 SMIRVGTNY GBM, SCLC 248 VTDIDELGK AML 249 GVGFTELEY HCC250 GYVCNACGLY BRCA 251 GIEMTYETY BRCA, CLL, SCLC 252 DTTSHTYLQY GBM 253YLESHGLAY GBM, HCC, MEL, PACA, SCLC 254 FLFNDALLY CCC, GBC, HNSCC, NHL,NSCLCadeno, NSCLCsquam, PACA 255 WELDSLEY BRCA, CCC, GBC, GC,HNSCC, MEL, NHL, NSCLCadeno, NSCLCsquam, OSCAR, PACA, UBC, UEC 256HAFESNNFIY CCC, GBC, NSCLCadeno 257 KSEMNVNMKY CCC, GBC, NSCLCadeno 258RPSSVLTIY GBM, HCC, HNSCC, NSCLCadeno, NSCLCsquam, OSCAR, SCLC, UBC 259APDEVVALL BRCA, CCC, MEL, OC, UEC 260 KPTEDSANVY GBM 261 MTEGSTVNTEYCCC, GBC, MEL, OC, RCC, SCLC, UEC 262 NVKHFLNDLY CCC, GBC, MEL,OC, RCC, SCLC, UEC 263 DCMDTEGSYM GBM, NSCLCother, OC, SCLC 264YRDPVFVSL CRC 265 LSDIDSRYI BRCA, CCC, GBC, HCC, HNSCC, MEL,NHL, OC, SCLC, UEC 266 LTDSFLLRF HCC 267 IVADDTVY PRCA 268 AILHHLYFYPRCA 269 LPSPAATIWDY CRC, GC, NSCLCadeno, PACA, RCC 270 DLKIDLAAQYHNSCC, NSCLCsquam, OSCAR 271 VAEPPVVCSY OC, SCLC 272 IPQDECLRYBRCA, GC, HCC, SCLC 273 CGPNEINHFY MEL, UBC 274 YADIHGDLL BRCA, CCC 275ESDEMENLLTY BRCA, PRCA 276 QITSFASGTSY AML 277 LPAPGFLFY BRCA 278AATVKSDIY BRCA, NHL, PRCA, SCLC 279 LMTVLLKY NSCLCother 280 TTEMVSNESVDYCCC, GBC, RCC 281 YPDLSELLM CCC, GBC, MEL, NHL, NSCLCother, OSCAR, SCLC,UBC 282 QAMPSWPTAAY MEL 283 ETILVSSSY HCC 284 TCSHTFVYY CCC, NSCLCother,NSCLCsquam, SCLC 285 VLPHHSEGACVY PRCA 286 ATDMEGNLNY GBM 287 ENSIEDLQYBRCA, PRCA, UEC 288 TEEKFVSY CRC, PACA, SCLC 289 YTSHEDIGY NSCLCother290 GQFTGTAGACRY OC 291 TSDVTGSLTY BRCA, CCC, GC, MEL, NHL, NSCLCother,NSCLCsquam, OC, RCC 292 VLDFAPPGASAY OC 293 IISVLIAIY SCLC 294 MMEMEGMYPRCA 295 GQRLDEAMISY OC, UBC 296 HMLAAMAY GBC, HCC, HNSCC, MEL,NSCLCadeno, NSCLCsquam, OC, OSCAR, UBC 297 RLDEAMISY OC, UBC 298KFDVINHYF GBC, HCC, HNSCC, MEL, NSCLCadeno, NSCLCsquam, OC, OSCAR, UBC299 EVDSVALSL GC, NHL, PACA 300 VSINPNSGDIY GBM, OC, UEC 301 ESQTCASDYCCC, GBC, GC, HNSCC, NSCLCsquam, OSCAR, UBC, UEC 302 FYLSTPENYHY MEL 303GFGGLSSQGVYY GC 304 FSENLIYTYI SCLC 305 YADLLIYTY HNSCC, OC, UBC, UEC306 KSFETTVRY MEL 307 DTDDRELRY GBM, SCLC 308 ELAAGQVVY GBM, SCLC 309EVDRNLIQY GBM, SCLC 310 KAFQELGVRY GBM, SCLC 311 TVTDGTHTDFY GBM, SCLC312 VTDGINPLI GBM, SCLC 313 VTDGTHTDFY GBM, SCLC 314 PPEANSLQGALY GBM315 VLKIELETY OC, UEC 316 YTCEECGQAF CLL 317 EDLLEVLDMY SCLC 318YMTSMALNY AML 319 FTDPHIITF CCC, NSCLCother, NSCLCsquam, SCLC 320QALQDKLQTFY CCC, SCLC 321 DGIADASNLSYY GBC, GC, PACA 322 FSELNPLALYBRCA, HNSCC, OC, OSCAR, UBC 323 KTLQKPVLPLY CLL 324 RTGIFPYRF CLL 325LQKPVLPLY CLL 326 STSRLTLFS PRCA 327 IMLSVDQHLY CCC 328 LLDEDNNIKLCRC, SCLC 329 NTDSMTLNNTAY AML, CLL, CRC, MEL, NHL 330 EGELSEGEHWY PRCA331 YLYQAPGSLALY BRCA, GC 332 SLISFKYTSY SCLC 333 LSDPQAELQFY GBM 334PSSMPECLSY CRC, NHL 335 PSSMPECLSYY CRC, NHL 336 ATNIQLNIDTY BRCA, CRC,GBC, GC, RCC 337 FTESNQYNIY UBC 338 YSPDSFNVSW MEL, OC 339 ESMDIFPLGWCLL 340 SVDSNLVAY PRCA 341 PANYLGKMTY PRCA 342 QTYMDGLLHY HNSCC, OSCAR343 YFGNYFTYY NSCLCother, SCLC 344 AVNALQSVY CRC, PRCA 345 NTMDAVPRIDHYCRC 346 VAGLEAGVLY SCLC 347 SADHPGLTF GBM 348 DSTDGCLLSF MEL 349HLLSVSLYY HCC 350 LTDPQVSYV MEL 351 VLDPMLDFY OC, UEC 352 YPVVVAESMYOC, UEC 353 RLNGSVASY OC, UEC 354 EIIRYIFAY AML 355 MADRGEARL GBC, NHL356 NSENHILKY HNSCC, SCLC 357 MSPDIALLYL NSCLCother 358 YMSPDIALLYNSCLCother 359 NKEINYFMY HCC 360 RFDDINQEF HNSCC, OSCAR 361 FTAEEGQLYHCC, PRCA 362 SGALDEAAAY HCC, PRCA 363 LTDRDVSFY HCC 364 DTGYLQLYYHNSCC, OC, OSCAR, UBC 365 FVDTKVPEH BRCA 366 ITVDVRDEF HNSCC, OC,OSCAR, UBC 367 LTDTGYLQLY HNSCC, OC, OSCAR, UBC 368 ESAATGQLDY GBM 369AVMEAAFVY PRCA 370 RLSTIRHLY BRCA, GBC, GC, NSCLCadeno, NSCLCsquam,OSCAR, PACA 371 WSDSTSQTIY HNSCC 372 SRSDFEWVY BRCA, GC, UBC 373FHADSDDESF CRC 374 LTSVVVTLW AML 375 ASSLDSLHY BRCA 376 EDDEDEDLY CLL377 YADPSANRDLL GBM 378 TAKAPSTEY CRC, GC, NSCLCadeno, PACA 379SLIIDDTEY GC, PACA, UBC, UEC 380 VACGNNPVY GC, PACA, UBC, UEC 381ETSFSTSHY HNSCC, OSCAR 382 YEPATMEQY MEL 383 PPDHAVGRTKY PRCA 384RFRSITQSYY BRCA, CLL 385 SANALILTY BRCA, CLL 386 NSALNPLLY NSCLCadeno,RCC UEC 387 LMEKEDYHSLY MEL 388 YTAHVGYSMY UEC 389 YYDLVESTF AML 390FSEPFHLIVSY MEL CLL, NHL 391 GSNPARYEF BRCA, CRC, GC, HCC, GBC, HNSCC,UEC NSCLCadeno MEL, NSCLCsquam, OC, OSCAR, SCLC, UBC 392 TQHFVQENYCRC, NHL, PACA GBC, GC, HCC, NSCLCadeno, HNSCC, MEL, UEC NSCLCsquam,OSCAR, SCLC, UBC 393 QVWGGQPVY MEL 394 QVPLDCVLY MEL 395 ILKGGSGTY MEL396 LPDPNVQKY NHL 397 NSAINPLIY CRC, GBC, GC 398 YYYDTHTNTY CRC

Example 3

In Vitro Immunogenicity for MHC Class I Presented Peptides

In order to obtain information regarding the immunogenicity of theTUMAPs of the present invention, the inventors performed investigationsusing an in vitro T-cell priming assay based on repeated stimulations ofCD8+ T cells with artificial antigen presenting cells (aAPCs) loadedwith peptide/MHC complexes and anti-CD28 antibody. This way theinventors could show immunogenicity for HLA-A*01 restricted TUMAPs ofthe invention, demonstrating that these peptides are T-cell epitopesagainst which CD8+ precursor T cells exist in humans (Table 10).

In Vitro Priming of CD8+ T Cells

In order to perform in vitro stimulations by artificial antigenpresenting cells loaded with peptide-MHC complex (pMHC) and anti-CD28antibody, the inventors first isolated CD8+ T cells from fresh HLA-A*02leukapheresis products via positive selection using CD8 microbeads(Miltenyi Biotec, Bergisch-Gladbach, Germany) of healthy donors obtainedfrom the University clinics Mannheim, Germany, after informed consent.

PBMCs and isolated CD8+ lymphocytes were incubated in T-cell medium(TCM) until use consisting of RPMI-Glutamax (Invitrogen, Karlsruhe,Germany) supplemented with 10% heat inactivated human AB serum(PAN-Biotech, Aidenbach, Germany), 100 U/ml Penicillin/100 μg/mlStreptomycin (Cambrex, Cologne, Germany), 1 mM sodium pyruvate (CC Pro,Oberdorla, Germany), 20 μg/ml Gentamycin (Cambrex). 2.5 ng/ml IL-7(PromoCell, Heidelberg, Germany) and 10 U/ml IL-2 (Novartis Pharma,Nurnberg, Germany) were also added to the TCM at this step.

Generation of pMHC/anti-CD28 coated beads, T-cell stimulations andreadout was performed in a highly defined in vitro system using fourdifferent pMHC molecules per stimulation condition and 8 different pMHCmolecules per readout condition.

The purified co-stimulatory mouse IgG2a anti human CD28 Ab 9.3 (Jung etal., 1987) was chemically biotinylated usingSulfo-N-hydroxysuccinimidobiotin as recommended by the manufacturer(Perbio, Bonn, Germany). Beads used were 5.6 μm diameter streptavidincoated polystyrene particles (Bangs Laboratories, Illinois, USA).

pMHC used for positive and negative control stimulations wereA*0201/MLA-001 (peptide ELAGIGILTV (SEQ ID NO. 433) from modifiedMelan-A/MART-1) and A*0201/DDX5-001 (YLLPAIVHI from DDX5, SEQ ID NO.434), respectively.

800.000 beads/200 μl were coated in 96-well plates in the presence of4×12.5 ng different biotin-pMHC, washed and 600 ng biotin anti-CD28 wereadded subsequently in a volume of 200 μl. Stimulations were initiated in96-well plates by co-incubating 1×10⁶ CD8+ T cells with 2×10⁶ washedcoated beads in 200 μl TCM supplemented with 5 ng/ml IL-12 (PromoCell)for 3 days at 37° C. Half of the medium was then exchanged by fresh TCMsupplemented with 80 U/ml IL-2 and incubating was continued for 4 daysat 37° C. This stimulation cycle was performed for a total of threetimes. For the pMHC multimer readout using 8 different pMHC moleculesper condition, a two-dimensional combinatorial coding approach was usedas previously described (Andersen et al., 2012) with minor modificationsencompassing coupling to 5 different fluorochromes. Finally, multimericanalyses were performed by staining the cells with Live/dead near IR dye(Invitrogen, Karlsruhe, Germany), CD8-FITC antibody clone SK1 (BD,Heidelberg, Germany) and fluorescent pMHC multimers. For analysis, a BDLSRII SORP cytometer equipped with appropriate lasers and filters wasused. Peptide specific cells were calculated as percentage of total CD8+cells. Evaluation of multimeric analysis was done using the FlowJosoftware (Tree Star, Oregon, USA). In vitro priming of specificmultimer+CD8+ lymphocytes was detected by comparing to negative controlstimulations. Immunogenicity for a given antigen was detected if atleast one evaluable in vitro stimulated well of one healthy donor wasfound to contain a specific CD8+ T-cell line after in vitro stimulation(i.e. this well contained at least 1% of specific multimer+ among CD8+T-cells and the percentage of specific multimer+ cells was at least 10×the median of the negative control stimulations).

In Vitro Immunogenicity

In vitro immunogenicity for acute myeloid leukemia, breast cancer,cholangiocellular carcinoma, chronic lymphocytic leukemia, colorectalcancer, gallbladder cancer, glioblastoma, gastric cancer, hepatocellularcarcinoma, head and neck squamous cell carcinoma, melanoma, non-Hodgkinlymphoma, lung cancer (including non-small cell lung canceradenocarcinoma, squamous cell non-small cell lung cancer, and small celllung cancer), ovarian cancer, esophageal cancer, pancreatic cancer,prostate cancer, renal cell carcinoma, urinary bladder carcinoma,uterine and endometrial cancer peptides was examined. For tested HLAclass I peptides, in vitro immunogenicity could be demonstrated bygeneration of peptide specific T-cell lines. Exemplary flow cytometryresults after TUMAP-specific multimer staining for two peptides of theinvention are shown in FIGS. 2A-2P and 3A-3G together with correspondingnegative controls. Results for 13 peptides from the invention aresummarized in Table 10a and results for further 28 peptides from theinvention are summarized in Table 10b.

TABLE 10a in vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% =++; 50%-69% = +++; >= 70% = ++++ Seq ID No Sequence Wells positive [%]399 YVGKEHMFY + 400 NTDNNLAVY + 401 VWSNVTPLKF + 403 SADDIRGIQSLY + 404FVDNQYWRY + 408 VWSDVTPLTF + 414 LTEGHSGNYY + 415 VWSDVTPLNF + 417KLDRSVFTAY ++++ 420 VTDLEMPHY + 421 RSDPGGGGLAY + 427 LTDYINANY + 429VSDSECLSRY ++

TABLE 10b in vitro immunogenicity of HLA class I peptidesof the invention Exemplary results of in vitro immunogenicityexperiments conducted by the applicant for thepeptides of the invention. <20% = +; 20%-49% =++; 50%-69% = +++; >= 70% = ++++ SEQID Sequence Wells positive [%]  10VSERTGISY +  11 ASDHWRGRY +++  12 YTDFVGEGLY +  18 RSDPVSLRY ++  19LTEGHSGNY +  24 NLDHYTNAY +  26 ASDDFRSKY ++  27 PSEVPVDSHY +  33SMDPVTGYQY +  37 MTSTEQSLYY +  42 ASDVDTLLK +  53 YLEDRPLSQLY +  54EVDIHTIHY +  60 MTEKFLFLY +  61 SSDIVALGGFLY ++  64 TSEISQNALMYY +  70YTELVEEKY +  71 LTDSTTRTTY +  73 STDSASYY +  75 FTDYELKAY +  77FTSDTGLEY +++  79 ASDLEPRELLSY +  81 YSDLHTPGRY +  82 LTEKSHIRY +  83DTEFHGGLHY +  84 ESEMIKFASYY + 386 NSALNPLLY + 387 LMEKEDYHSLY +

Example 4

Synthesis of Peptides

All peptides were synthesized using standard and well-established solidphase peptide synthesis using the Fmoc-strategy. Identity and purity ofeach individual peptide have been determined by mass spectrometry andanalytical RP-HPLC. The peptides were obtained as white to off-whitelyophilizes (trifluoro acetate salt) in purities of >50%. All TUMAPs arepreferably administered as trifluoro-acetate salts or acetate salts,other salt-forms are also possible.

Example 5

MHC Binding Assays

Candidate peptides for T cell based therapies according to the presentinvention were further tested for their MHC binding capacity (affinity).The individual peptide-MHC complexes were produced by UV-ligandexchange, where a UV-sensitive peptide is cleaved upon UV-irradiationand exchanged with the peptide of interest as analyzed. Only peptidecandidates that can effectively bind and stabilize the peptide-receptiveMHC molecules prevent dissociation of the MHC complexes. To determinethe yield of the exchange reaction, an ELISA was performed based on thedetection of the light chain (β2m) of stabilized MHC complexes. Theassay was performed as generally described in Rodenko et al. (Rodenko etal., 2006)

96 well MAXISorp plates (NUNC) were coated over night with 2 ug/mlstreptavidin in PBS at room temperature, washed 4× and blocked for 1 hat 37° C. in 2% BSA containing blocking buffer. RefoldedHLA-A*02:01/MLA-001 monomers served as standards, covering the range of15-500 ng/ml. Peptide-MHC monomers of the UV-exchange reaction werediluted 100-fold in blocking buffer. Samples were incubated for 1 h at37° C., washed four times, incubated with 2 ug/ml HRP conjugatedanti-62m for 1 h at 37° C., washed again and detected with TMB solutionthat is stopped with NH₂SO₄. Absorption was measured at 450 nm.Candidate peptides that show a high exchange yield (preferably higherthan 50%, most preferred higher than 75%) are generally preferred for ageneration and production of antibodies or fragments thereof, and/or Tcell receptors or fragments thereof, as they show sufficient avidity tothe MHC molecules and prevent dissociation of the MHC complexes.

MHC:peptide binding results for 391 peptides from the invention aresummarized in Table 11.

TABLE 11 MHC class I binding scores. Binding of HLA-classI restricted peptides to HLA-A*01:01 was rangedby peptide exchange yield: >10% = +; >20% = ++; >50 = +++; >75% = ++++SEQID Sequence Peptide exchange   1 TLDSTRTLY ++++   2 VDPIGHLY ++   3FGTTPAAEYF ++   4 RIEAIRAEY ++   5 FMVIAGMPLFY +++   6 ARDPITFSF ++   7ASDDVRIEVGLY ++++   8 TSRAANIPGY +   9 QLDSTLDSY +++  10 VSERTGISY +++ 11 ASDHWRGRY +++  12 YTDFVGEGLY +++  13 NTHTGTRPY ++  14 QSEKEPGQQY +++ 15 YLDSSKPAVY +++  16 NSDISIPEY +++  17 ASWAVLCYY +++  18 RSDPVSLRY++++  19 LTEGHSGNY ++++  20 LSAQHRMLA ++  21 LSSAVNPIIY ++  22 VMDTLGLFY++++  23 DTDPLKAAGL +  24 NLDHYTNAY +++  25 AMMQEAQLAY +++  26 ASDDFRSKY+++  27 PSEVPVDSHY ++++  28 PSEVPVDSHYY +++  29 TLEDLDNLYNY +++  30VTTDKPRAY ++  31 VSDHLQAGMLGQY +++  32 GTDKQNSTLRY +++  33 SMDPVTGYQY+++  34 SSWSAGENDSY +++  35 SWSAGENDSYS +  36 MTSTEQSLY +++  37MTSTEQSLYY +++  38 KSWSQSSSLMY +++  39 WSQSSSLMY ++++  40 TSDQLGYSY +++ 41 HSDLLEDSKY +++  42 ASDVDTLLK +++  43 ETEPERHLGSY +++  44 IPSFNEMVY++  45 NLDPNKIY +  46 RSDPGGGGLAYAAY ++++  47 WSDGVPLLY ++++  48FTTQDELLVY +++  49 GSFSIQHTY ++  50 ITDEDEDMLSY +++  51 STEERRLNY +++ 52 TTQDELLVY +++  53 YLEDRPLSQLY ++++  54 EVDIHTIHY +++  55 ATEGDVLNY+++  56 VTEYAEEIYQY +++  57 ASDPASSTSCY +++  58 YLENSASWY ++++  59FTDSQGNDIK +++  60 MTEKFLFLY ++++  61 SSDIVALGGFLY +++  62 VSELVTTGHY+++  63 TSEISQNALMY ++++  64 TSEISQNALMYY +++  65 SSDFDPLVY ++++  67NVDQNQNSY +++  68 QSLPEFGLTY ++  69 QSLPEFGLTYY ++  70 YTELVEEKY +++  71LTDSTTRTTY +++  72 VTDSTTKIAY +++  73 STDSASYY ++++  74 EMEQQSQEY ++++ 75 FTDYELKAY ++++  76 QTDVERIKDTY +++  77 FTSDTGLEY ++++  78 QLDSAVKNLY+++  79 ASDLEPRELLSY +++  80 ELCPLPGTSAY ++  81 YSDLHTPGRY +++  82LTEKSHIRY +++  83 DTEFHGGLHY +++  84 ESEMIKFASYY ++++  85 SSDNYEHWLY++++  86 VDPASNTY ++  87 AFDDIATYF ++  88 KEVDPAGHSYI ++  89 EVYDGREHSAY++  90 YEDHFPLLF ++  92 TTDDTTAMASAS ++  93 HLKILSPIY +++  94 KPSAVKDSIY++  95 SSDPKAVMF ++  97 FPAPPAHWFY ++  98 NFSDLVFTY ++  99 AADSNPSEL ++100 TTSSAISWILY +++ 101 SITDVDFIY ++ 102 STIRGELFF ++ 103 ITDTLIHLM +++104 ITDTLIHL ++ 105 VVFDKSDLAKY ++ 106 EVVEGKEWGSFY +++ 107 TTENSGNYY++++ 108 NSNLKFLEV ++ 109 ISEDKSISF ++ 110 IGDKVDAVY ++ 111 TPIPFDKILY++ 112 KASSVSAEDGY ++ 113 ASCRSSAEY ++++ 114 AVAAAAGASLY ++ 115NEIDIHSIYFY ++ 116 RSDIGEFEW ++ 117 SPAKQFNIY +++ 118 LTWAHSAKY +++ 119TVFDENLSRY ++ 120 LVDENQSWY +++ 121 SADEAHGLL ++ 122 ISEAPLTEV ++ 123LLKAKDILY ++ 124 FLKVTGYDKDDY ++ 125 FQYELRELY ++++ 126 TTDPKKFQY ++++127 VPFNLITEY + 128 YTEFVDATFTK ++++ 129 STIDFRAGF ++ 130 YIGLKGLYF ++131 LEDGIEQSAY ++ 132 RTHIGYKVY +++ 133 ITDVGPGNY +++ 134 SAPSSSGSPLY ++135 TFDKQIVLL ++ 136 RRLNFSGFGY +++ 137 EAYLERIGY +++ 138 IPVHDSVGVTY+++ 139 PVHDSVGVTY ++ 140 SQHIFTVSY ++ 141 DAVAPGREY ++ 142 IEKFAVLY +++143 HVSGQMLYF ++ 144 RTIEGDFLW ++ 145 LSDAVHVEF +++ 146 LCATVCGTEQY ++147 AQVQDTGRY +++ 148 GTKQWVHARY + 149 PIMSSSQALY ++ 150 FTTLSDLQTNMA ++151 YEVDTKLLSL ++ 152 YLEDRPLSQ ++ 153 HSIEVFTHY ++ 154 SIEVFTHY +++ 155HTMEVTVY +++ 156 STALSILLL ++ 157 GLIEVVTGY ++++ 158 EVTDRNMLAF +++ 159RQAPGPARDY ++ 160 EVLGEEMYAY ++ 161 EAAPDIMHY ++ 162 IADNPQLSFY ++++ 163KIRAEVLSHY ++ 164 KLAGTVFQY ++ 165 VSVYNSYPY ++ 166 YHRICELLSDY ++++ 167RAVQPGETY +++ 168 VQPGETYTY +++ 169 TVDNANILL ++ 170 VQIAKGMNY +++ 171ITDFGLAKL ++ 172 FSEPFHLIV +++ 173 QSTTGVSHY +++ 174 TSEVEGLAFVSY +++175 GLEYEAPKLY ++ 176 HTDLESPSAVY +++ 177 LVDGKWQEF ++ 178 TQRTSFQFY ++179 SSTDFTFASW + 180 AQISDTGRY ++ 181 SVTDLIGGKW ++ 182 TQPELSSRY ++ 183LADTDLGMTF ++ 184 KTIQEVAGY ++ 185 NSDESADSEPHKY ++++ 186 AVSSGLFFY ++187 TQKSVQVLAY ++ 188 DIPDYLLQY +++ 189 FRGVFVHRY ++ 190 VSSTVHNLY ++++191 FTRAFDQLRM +++ 192 LAFYYGMY ++ 193 SQNGQLIHY +++ 194 CYTADNEMGY +++195 YTADNEMGYY +++ 196 RLAQYTIERY ++ 197 NDEIDKLTGY ++++ 198 KLTDYINANY++ 199 LCAAVLAKY ++ 200 SLPEFGLTY ++ 201 SLPEFGLTYY ++ 202 QTDINGGSLK+++ 203 LSQDELSKF ++ 204 NVKEAPTEY ++ 205 RMQEGSEVY ++ 207 LLEGEDAHLTQY+++ 208 LLISKAEDY ++ 209 EADPFLKYL ++ 210 LLEADPFLKY ++++ 211 YLNEWGSRF++ 212 MMTDLTSVY +++ 213 VSDSTTEITY +++ 214 VQDPSLPVY ++ 215 DTLEAATSLY++ 216 NSMLDPLVY ++++ 217 LMDEGAVLTL ++ 218 FTAQLQLY +++ 219 KTELETALYY+++ 220 DVERIKDTY ++ 221 TDVERIKDTY +++ 222 GSPDAVVSY + 223 NAVDVVPSSF +224 RTDEGDNRVW ++ 225 STDPNIVRK +++ 226 QITPKHNGLY ++ 227 ESAPKEVSRY ++228 KSFDDIAKY ++ 229 MTDVFIDY +++ 230 CVIETFHKY +++ 231 LLPLLVMAY ++ 232RYLNIVHATQLY ++ 233 RINSATGQY ++ 234 YTDLTTIQV ++++ 235 SIEIDHTQY ++ 236VLDSLLAQY ++++ 237 AQEAAVFLTLY +++ 238 ETDWGLLKGHTY ++++ 239 SSERGSPIEKY+++ 240 EVLDSLLAQY ++ 241 SLMVASLTY ++ 242 GTNLPTLLW + 243 LTSEDTGAY +++244 VTKYIAGPY ++ 245 LSDNAANRY +++ 246 ARLEGEIATY ++ 247 SMIRVGTNY ++248 VTDIDELGK +++ 249 GVGFTELEY +++ 250 GYVCNACGLY ++ 251 GIEMTYETY ++252 DTTSHTYLQY ++ 253 YLESHGLAY ++++ 254 FLFNDALLY ++++ 255 WELDSLEY ++256 HAFESNNFIY ++ 257 KSEMNVNMKY ++++ 258 RPSSVLTIY +++ 259 APDEVVALL ++260 KPTEDSANVY +++ 261 MTEGSTVNTEY ++++ 262 NVKHFLNDLY +++ 263DCMDTEGSYM ++ 264 YRDPVFVSL ++ 265 LSDIDSRYI ++ 266 LTDSFLLRF ++++ 267IVADDTVY +++ 268 AILHHLYFY + 269 LPSPAATIWDY +++ 270 DLKIDLAAQY ++ 271VAEPPVVCSY ++ 272 IPQDECLRY +++ 273 CGPNEINHFY ++ 274 YADIHGDLL +++ 275ESDEMENLLTY +++ 276 QITSFASGTSY +++ 277 LPAPGFLFY ++ 278 AATVKSDIY ++279 LMTVLLKY +++ 280 TTEMVSNESVDY ++++ 281 YPDLSELLM ++ 282 QAMPSWPTAAY++ 283 ETILVSSSY ++ 284 TCSHTFVYY ++ 285 VLPHHSEGACVY +++ 286 ATDMEGNLNY+++ 287 ENSIEDLQY +++ 288 TEEKFVSY + 289 YTSHEDIGY ++++ 290 GQFTGTAGACRY++ 291 TSDVTGSLTY ++++ 292 VLDFAPPGASAY ++++ 294 MMEMEGMY +++ 295GQRLDEAMISY +++ 296 HMLAAMAY ++ 297 RLDEAMISY +++ 298 KFDVINHYF ++ 299EVDSVALSL ++ 300 VSINPNSGDIY +++ 301 ESQTCASDY +++ 302 FYLSTPENYHY +++303 GFGGLSSQGVYY +++ 304 FSENLIYTYI ++ 305 YADLLIYTY ++++ 306 KSFETTVRY++ 307 DTDDRELRY +++ 308 ELAAGQVVY + 309 EVDRNLIQY +++ 310 KAFQELGVRY ++311 TVTDGTHTDFY ++ 312 VTDGINPLI ++ 313 VTDGTHTDFY +++ 314 PPEANSLQGALY++ 315 VLKIELETY +++ 316 YTCEECGQAF ++ 317 EDLLEVLDMY ++ 318 YMTSMALNY+++ 319 FTDPHIITF ++++ 320 QALQDKLQTFY ++ 321 DGIADASNLSYY ++ 322FSELNPLALY ++++ 323 KTLQKPVLPLY ++ 324 RTGIFPYRF ++ 325 LQKPVLPLY ++ 326STSRLTLFS ++ 327 IMLSVDQHLY +++ 328 LLDEDNNIKL ++ 329 NTDSMTLNNTAY ++++330 EGELSEGEHWY +++ 331 YLYQAPGSLALY ++++ 333 LSDPQAELQFY ++++ 334PSSMPECLSY ++++ 335 PSSMPECLSYY +++ 336 ATNIQLNIDTY +++ 337 FTESNQYNIY++++ 338 YSPDSFNVSW ++ 339 ESMDIFPLGW ++ 340 SVDSNLVAY +++ 341PANYLGKMTY +++ 342 QTYMDGLLHY ++++ 343 YFGNYFTYY + 344 AVNALQSVY ++ 345NTMDAVPRIDHY ++++ 346 VAGLEAGVLY ++ 347 SADHPGLTF ++ 348 DSTDGCLLSF ++349 HLLSVSLYY + 350 LTDPQVSYV +++ 351 VLDPMLDFY ++++ 352 YPVVVAESMY ++353 RLNGSVASY ++ 354 EIIRYIFAY ++ 355 MADRGEARL ++ 356 NSENHILKY ++++357 MSPDIALLYL +++ 358 YMSPDIALLY ++++ 359 NKEINYFMY +++ 360 RFDDINQEF +361 FTAEEGQLY +++ 362 SGALDEAAAY ++ 363 LTDRDVSFY ++++ 364 DTGYLQLYY++++ 365 FVDTKVPEH ++ 366 ITVDVRDEF + 367 LTDTGYLQLY ++++ 368 ESAATGQLDY+++ 369 AVMEAAFVY +++ 370 RLSTIRHLY +++ 371 WSDSTSQTIY ++++ 372SRSDFEWVY ++ 373 FHADSDDESF + 375 ASSLDSLHY ++++ 376 EDDEDEDLY +++ 377YADPSANRDLL ++ 378 TAKAPSTEY ++ 379 SLIIDDTEY ++ 380 VACGNNPVY +++ 381ETSFSTSHY ++++ 382 YEPATMEQY ++ 383 PPDHAVGRTKY ++ 384 RFRSITQSYY + 385SANALILTY ++ 386 NSALNPLLY ++++ 387 LMEKEDYHSLY +++ 388 YTAHVGYSMY ++++389 YYDLVESTF + 390 FSEPFHLIVSY ++++ 391 GSNPARYEF ++ 392 TQHFVQENY ++393 QVWGGQPVY + 394 QVPLDCVLY ++ 395 ILKGGSGTY ++ 396 LPDPNVQKY ++ 397NSAINPLIY ++++ 398 YYYDTHTNTY +

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1. A method of treating a patient who has cancer, comprisingadministering to said patient a population of activated T cells that arecapable of killing cancer cells that present a peptide consisting of theamino acid sequence of SEQ ID NO: 361, wherein said cancer is selectedfrom breast cancer, gallbladder cancer, glioblastoma, hepatocellularcarcinoma, non-small cell lung cancer, pancreatic cancer, prostatecancer, renal cell carcinoma, small cell lung cancer, urinary bladdercarcinoma, and uterine and endometrial cancer.
 2. The method of claim 1,wherein the T cells are autologous to the patient.
 3. The method ofclaim 1, wherein the T cells are obtained from a healthy donor.
 4. Themethod of claim 1, wherein the T cells are obtained from tumorinfiltrating lymphocytes or peripheral blood mononuclear cells.
 5. Themethod of claim 1, further comprising administering to said patient anadjuvant selected from anti-CD40 antibody, imiquimod, resiquimod,GM-CSF, cyclophosphamide, sunitinib, bevacizumab, interferon-alpha,interferon-beta, CpG oligonucleotides and derivatives, poly-(I:C) andderivatives, RNA, sildenafil, particulate formulations with poly(lactideco-glycolide) (PLG), virosomes, interleukin (IL)-1, IL-2, IL-4, IL-7,IL-12, IL-13, IL-15, IL-21, and IL-23.
 6. The method of claim 1, whereinthe activated T cells are cytotoxic T cells produced by contacting Tcells with an antigen presenting cell that expresses the peptide in acomplex with an MEW class I molecule on the surface of the antigenpresenting cell, for a period of time sufficient to activate said Tcell.
 7. The method of claim 1, wherein the cancer is breast cancer. 8.The method of claim 1, wherein the cancer is gallbladder cancer.
 9. Themethod of claim 1, wherein the cancer is glioblastoma.
 10. The method ofclaim 1, wherein the cancer is hepatocellular carcinoma.
 11. The methodof claim 1, wherein the cancer is non-small cell lung cancer.
 12. Themethod of claim 1, wherein the cancer is pancreatic cancer.
 13. Themethod of claim 1, wherein the cancer is prostate cancer.
 14. The methodof claim 1, wherein the cancer is renal cell carcinoma.
 15. The methodof claim 1, wherein the cancer is small cell lung cancer.
 16. The methodof claim 1, wherein the cancer is urinary bladder carcinoma.
 17. Themethod of claim 1, wherein the cancer is uterine and endometrial cancer.18. The method of claim 5, wherein the adjuvant comprises IL-7.
 19. Themethod of claim 5, wherein the adjuvant comprises IL-15.
 20. The methodof claim 5, wherein the adjuvant comprises IL-21.